Substituted bithiophenes and dithienylpyrroles

ABSTRACT

Electrically conducting homo- and/or copolymers and/or tripolymers can be produced from novel monomers, such as a 3-substituted 2,5-di(2-thienyl)pyrrole. The polymers exhibit unexpectedly high stability and conductivities, and can be functionalized, such as with an enzyme, like glucose oxidase, or an ion-specific binding site, like a crown ether, or an antigen, without adversely affecting the conductivity of the polymer. The functionalized, conducting polymer can be used in a diagnostic device to determine the presence and concentration of a specific analyte in a liquid medium. For example, the presence and concentration of glucose is determined by measuring the conductivity change in the polymer caused by the vibrational excitation induced in the enzyme, glucose oxidase, from its reaction with the glucose and/or by measuring a secondary effect of the enzyme/substrate reaction, such as the change in the conductivity of the conducting polymer caused by the generation of hydrogen peroxide during the glucose-glucose oxidase reaction.

This is a division of application Ser. No. 114,011, filed Oct. 29, 1987,now U.S. Pat. No. 4,886,625.

FIELD OF THE INVENTION

The present invention relates to a method of determining analyteconcentrations by utilizing analyte sensors that employ conductingorganic polymers. More particularly, conducting polymers, synthesizedfrom novel monomers, can be covalently functionalized with an enzyme,antigen or an ion specific binding site, and employed in a diagnosticdevice to selectively assay a liquid medium for the presence andconcentration of a specific analyte. The presence and concentration ofthe specific analyte is determined by measuring the change inconductivity of the polymer arising either from transduction of thevibrational excitation induced in the covalently-bound functionality bythe reaction of the functionality with the analyte, and/or by measuringthe change in conductivity of the polymer arising from secondary effectsof the reaction between the covalently-bound functionality and theanalyte, such as the generation of hydrogen peroxide. Surprisingly andunexpectedly, the monomers utilized to prepare the organic conductingpolymers of the present invention yield polymers having a high degree ofstability and conductivity. The monomers, each having a five-memberedheteroaromatic ring substituted in the three position, provide polymershaving unexpectedly high conductivities compared to prior art conductingpolymers prepared from functionalized five membered heteroaromatic ringcompounds. Even more surprisingly, this high degree of polymerconductivity is maintained after functionalization of the polymer withan enzyme, antigen or ion-specific binding site. As a result,functionalized conducting polymers are available for use in diagnosticdevices to determine analyte concentrations in liquid media.

BACKGROUND OF THE INVENTION

Investigators have shown an intense interest in organic conductingpolymers that can be synthesized chemically, like polyacetylene, orelectrochemically, like polypyrrole and polythiophene. The organicconducting polymers have several potential applications in the fields ofbatteries, display devices, corrosion prevention in metals andsemiconductors and in microelectronic devices such as diodes,transistors, sensors, light emitting devices and energy conversion andstorage elements. However, present day organic conducting polymerspossess several limitations that have hindered the expansion of organicconducting polymers into these and other potential application areas.The limitations found in the three most extensively studied conductingpolymers, polyacetylene, polypyrrole and polythiophene, illustrate thegeneral problems encountered by investigators in the field of conductingpolymers and why the use of conducting polymers has been impeded.

For example, polyacetylene, among the first organic conducting polymers,is prepared chemically from acetylene by using an appropriate catalyst.As prepared chemically, polyacetylene is an insulator, exhibitingconductivities in the range of 10⁻¹⁰ S/cm to 10⁻¹³ S/cm (Siemens percentimeter) that correspond to the conductivity of known insulators,such as glass and DNA. However, polyacetylene can be doped using avariety of oxidizing or reducing agents, such as antimony pentafluoride,the halogens, astatine pentafluoride, or aluminum chloride. By doping,polyacetylene is converted into a highly conducting polymer, exhibitinga conductivity of approximately 10³ S/cm, therefore exhibiting theconductivity of metals such as bismuth. However, polyacetylene suffersfrom the drawbacks of extreme instability in air and a precipitous dropin conductivity whenever an acetylenic hydrogen is replaced by an alkylor other substituent group. Accordingly, the instability ofpolyacetylene in the presence of oxygen, and its inability to befunctionalized and maintain its high conductivity, makes thepolyacetylenes unsuitable conducting polymers for use as an analytesensor.

Polypyrrole, a conducting polymer similar to polyacetylene, can besynthesized chemically or electrochemically and exhibits conductivitiesranging from about 1 S/cm to about 100 S/cm. As will be discussed morefully hereinafter, conducting polypyrrole is a doped material,incorporating the anion of the supporting electrolyte. Polypyrrolehaving a molecular weight of up to approximately 40,000 has beensynthesized; however, conductivity is observed in polypyrrole containingas few as six monomer units. Normally, polypyrrole, and other conductingpolymers, are low molecular weight polymers containing less than 100monomer units.

Investigators have found that placing alkyl groups on either thenitrogen or the carbons of the heteroaromatic pyrrole ring decreases theconductivity of polypyrrole. For example, an unsubstituted polypyrrole,incorporating the tetrafluoroborate anion as the compensatingcounterion, exhibits a conductivity of 40 S/cm, whereas the N-methylderivative, incorporating the same dopant, exhibits a conductivity of10⁻³ S/cm; the three-methyl derivative of pyrrole exhibits aconductivity of 4 S/cm; 3,4-dimethyl derivative, a conductivity of 10S/cm; and the 3,4-diphenyl derivative, a conductivity of 10⁻³ S/cm.

The conductivity decrease in substituted polypyrroles is attributed toseveral factors. First, and of prime importance, the substituentintroduced onto the heteroaromatic pyrrole ring cannot alter theoxidation potential of the parent heteroaromatic to the extent thatelectropolymerization at the anode is precluded. Secondly, and a relatedconsideration, the aromatic pi-electron system of the parent heterocyclemust be maintained. Disruption of the pi-electron system of theheteroaromatic ring will adversely affect the relative stability of thearomatic and quinoid-like forms, illustrated as structures I and II,respectively, and therefore seriously reduce conductivity. A thirdcritical consideration is that the functionality introduced onto theparent heterocycle must not create steric demands that preclude theadoption of a planar configuration by the conducting polymer. ##STR1##

The requirement that the conducting polymer must maintain a planarconfiguration has seriously hindered development of functionalized,conducting polymers. Numerous N-alkyl and N-aryl derivatives ofpolypyrrole have been prepared and discussed in the literature. However,it was found that even the simplest of these N-substituted polypyrroles,poly-N-methylpyrrole, exhibits conductivities that are three orders ofmagnitude lower than unsubstituted polypyrrole films doped with the samecounterion. It is also possible to produce thin films of poly-N-arylpyrroles, wherein the phenyl group is further substituted in the paraposition. However, polymers produced from these N-aryl pyrrolesinvariably exhibit conductivities three or more orders of magnitude lessthan the parent unsubsituted pyrrole. Such low conductivities precludethe use of these substituted polypyrroles in the development of analytesensors.

The steric interactions introduced by the pyrrole ring substituents isimportant because of the mechanism of charge transport through theconducting polymer system. In one charge transport mechanism, electriccharge is conducted through the polymer chain itself because ofbipolaron structures that exist along the polymer chain. The bipolaronstructure, illustrated in structure III and confirmed from spectroscopicevidence obtained on polythiophene, are defects occurring in the polymerlattice wherein two dopant counterions, A⁻, from the supportingelectrolyte, balance two positive centers found in the polymer. ##STR2##

Generally, the two positive centers are spaced, and confined, byapproximately four monomer units and these defects serve to transportcharge along the polymer chain. However, in order to transport chargealong the chain, compositions having the structures I, II and/or IIImust be planar, such that the charge can be transported along the planarpi-electron system of the chain. As can be seen in structure IV, if thesubstituents R and/or R' are sufficiently large, the steric interactionbetween R and R' can distort the pyrrole monomer units out of planarity,therefore destroying the planarity of the pi-electron system, anddestroying, or seriously reducing, the conductivity of the polymer. Asillustrated by the large conductivity drop in polypyrroles havingsubstituents positioned on the pyrrole ring, even substituents as smallas a methyl group introduce steric interactions sufficient toessentially destroy the conductivity of the polymer.

It also should be noted that investigators have found that R and/or R'substituents in structure IV should not be strongly electron-withdrawingor strongly electron-donating, as strong electronic effects also canserve to destroy the conductivity of the polymer. However, it has beenfound, especially for N-substituted pyrroles, that steric interactions,not electronic effects, are the main factor in determiningpolymerizability, polymer conductivity, and cyclic stability of thepolymer between the doped and undoped state. Steric interactions inpolythiophene derivatives are somewhat less dominant than those observedin polypyrrole derivatives. Steric interactions in polypyrrolederivatives are more dominant because the predominant destabilizinginteractions in pyrrole derivatives involve the hydrogen atom of thepyrrole nitrogen. These steric interactions are avoided inpolythiophenes. As a result, electronic effects play a more central rolein polythiophene derivatives.

Conducting organic polymers generally are amorphous, disorderedmaterials, and as a result, if bulk conductivity is to be sustained,charge transport must occur between polymer strands as well as alongsingle polymer strands. The probability of the interchain chargetransport is directly related to the distance between chains. Thedistance between polymer chains is acutely sensitive to, and dependentupon, two factors, the nature and size of the dopant counterion and thecharacter and steric requirements of the R and R' substituents ofstructure IV. This steric requirement imposes a significant constrainton the design of functionalized conducting polymers.

The synthesis and conductivities of polypyrrole and substitutedpolypyrroles have been extensively investigated as seen in the generalreferences cited below. These references include the informationdiscussed above and general information concerning the polypyrroles,such as that the specific dopant (A⁻) in structure III can seriouslyaffect the conductivity of the polymer; that conductivity is observedonly for alpha-alpha coupling of monomers and not for alpha-betacoupling of monomers (see structure V); and that polypyrrole films arestable, insoluble, and inert to most reagents, except possibly treatmentby alkalis. The conductivity and stability of polypyrrole makespolypyrrole a good candidate for use in analyte sensors, if thepolypyrrole conductivity can be maintained when functional groups areintroduced onto the heteroaromatic ring. ##STR3##

The representative references discussing the polypyrroles include:

G. Bidan, Tet. Lett. 26(6), 735-6 (1985).

P. Audebert, G. Bidan, an M. Lapowski, J. C. S. Chem. Comm., 887 (1986).

M. S. Wrighton, Science 231, 32 (1986).

R. A. Simon, A. I. Ricco and M. S. Wrighton, J. Am. Chem. Soc, 104, 2034(1982).

A. F. Diaz, J. Castillo, K. K. Kanazawa, J. A. Logan, M. Salmon and O.Fojards, J. Electroanal. Chem. 133, 233 (1982).

M. Saloma, M. Aguilar and M. Salmon, J. Electrochem. Soc. 132, 2379(1985).

M. V. Rosenthal, T. A. Skotheim, A. Melo, M. I. Florit, and M. Salmon,J. Electroanal. Chem. and Interfac. Chem. 1, 297 (1985).

G. Bidan and M. Guglielmi, Synth. Met. 15, 51 (1986).

M. Salmon and G. Bidan, J. Electrochem. Soc., 1897 (1985).

E. M. Genies and A. A. Syed, Synth. Met. 10, 27 (1984/85).

G. Bidan, A. Deronzier and J. C. Moutet, Nouveau Jour. de Chimie 8, 501(1984).

J. P. Travers, P. Audebert and G. Bidan, Mol. Cryst. Liq. Cryst. 118,149 (1985).

Another well-studied conducting polymer is polythiophene, whereinthiophene (structure V, X=S) is electrochemically polymerized to yield astable conducting polymer. Similarly, furan (structure V, X=O) alsoyields a stable conducting polymer similar to polypyrrole andpolythiophene. Polythiophene resembles polypyrrole in that polythiophenecan be cyclized between its conducting (oxidized) state and itsnonconducting (neutral) state without significant chemical decompositionof the polymer and without appreciable degradation of the physicalproperties of the polymer. Polythiophene, like polypyrrole, exhibitsconductivity changes in response both to the amount of dopant and to thespecific dopant, such as perchlorate, tetrafluoroborate,hexafluorophosphate, hydrogen sulfate, hexafluoroarsenate andtrifluoromethylsulfonate.

Substituents placed on the heteroaromatic thiophene ring can affect theresulting conducting polymer. For example, thiophene polymerization canbe affected by large substituents at the 3 and 4 positions, as seen inthe inability of 3,4-dibromothiophene to polymerize. The electronic andsteric effects introduced by the 3,4-dibromo substituents may preventchain propagation. However, in contrast to pyrrole, ring substituents onthiophene do not seriously reduce the conductivity of the resultingheteroaromatic polymer. For example, it has been found that for3-methylthiophene and 3,4-dimethylthiophene, the resulting substitutedpolythiophene exhibited an improved conductivity compared to the parentpolythiophene, presumably due to enhanced order in the polymer chain ofthe substituted thiophene. However, the methyl group is not a suitablesubstituent for the subsequent polymer surface functionalization neededto produce an analyte sensor.

The following are representative references concerning the synthesis andconductivity of polythiophene and substituted polythiophenes:

G. Tourillon, "Handbook of Conducting Polymers," T. A. Skotheim, ed.,Marcel Dekker, Inc., New York, 1986, p. 293.

R. J. Waltham, J. Bargon and A. F. Diaz, J. Phys. Chem. 87, 1459 (1983).

G. Tourillon and F. Garnier, J. Polym. Sci. Polym. Phys. Ed. 22, 33(1984).

G. Tourillon and F. Garnier, J. Electroanal. Chem. 161, 51 (1984).

A. F. Diaz and J. Bargon, "Handbook of Conducting Polymers," T. A.Skotheim, ed., Marcel Dekker, Inc., New York 1986, p. 81.

J. Bargon, S. Mohmand and R. J. Waltman, IBM, J. Res. Dev. 27, 330(1983).

G. Tourillon and F. Garnier, J. Phys. Chem. 87, 2289 (1983).

A. Czerwinski, H. Zimmer, C. H. Pham, and H. B. Mark, Jr., J.Electrochem. Soc. 132, 2669 (1985).

From the studies on the polyacetylenes, polypyrroles and polythiophenes,and from related studies on other conducting polymers, includingpolyparaphenylene, polyazulene, polycarbazole, polypyrene, polyanilineand polytriphenylene, it is apparent that a delicate balance existsbetween the electronic effects and the steric effects introduced by thesubstituents that renders a polymer of a substituted five or six memberheteroaromatic ring more conducting or less conducting than theunsubstituted parent heteroaromatic compound. Therefore, it would beadvantageous to develop a monomer that can be readily polymerized,chemically or electrochemically, to yield a conducting polymer havingsufficient conductivity such that the polymer can be used as an analytesensor in a diagnostic device to determine the presence andconcentration of an analyte in liquid media.

It is also apparent that a functionalized conducting polymer is requiredfor ultimate use as an analyte sensor. The polymer must not only possesssufficient conductivity, but the polymer also must contain moieties thatcan interact with the analyte of interest. This interaction then mustsufficiently alter the conductivity of the polymer in order tomeasurably detect the conductivity difference and convert thisconductivity change into an analyte concentration. It is to such aconducting polymer that the method of the present invention is directed.

The prior art does not include any known references to the method of thepresent invention. The prior art chemical modifications to conductingpolymers were unconcerned with the retention of high conductivity. Forexample, M. S. Wrighton et al, in the references cited above, havedeveloped N-alkylpyrroles in an attempt to improve the binding of apolymer film to a platinum electrode. In this study, only a very thinlayer of functionalized polypyrrole in contact with the electrode isrequired, therefore making the conductivity of the essentially monolayerfilm unimportant.

Saloma et al (J. Electrochem. Soc. 32, 2379 (1985)) have attempted tofunctionalize polymer films in order to modify electrode properties.Saloma et al attempted to utilize the conductivity of the functionalizedpolymer as an electronic mediator for any chemical effects occurring onthe attached moiety. However, this particular research area has beenbypassed by similar chemical modifications of metal electrodes (R. W.Murray, Acc. Chem. Res. 13, 135 (1980)).

M. V. Rosenthal et al disclosed, in M. V. Rosenthal, T. A. Skotheim, C.Linkous and M. I. Florit, Polym. Preprints 25, 258 (1984) and in M. V.Rosenthal, T. A. Skotheim, J. Chem. Soc. Chem. Commun 6, 342 (1985), anattempt to derivatize polypyrrole after polymerization.

The above referenced prior art concerning substituted pyrrole andsubstituted thiophene polymers is not directed to preparing conductingpolymers for use as an analyte sensor in a diagnostic device. Forexample, films prepared from the methyl derivative of thiophene were notsynthesized in order to attempt subsequent polymer surfacefunctionalization, but rather to prevent monomeric couplings through thebeta positions in order to introduce greater order, and thereforegreater conductivity, into the polymer. In the referenced prior art, theinvestigators attempted to characterize and improve polymer properties,as opposed to chemically utilizing the substituents on theheteroaromatic ring.

During the course of the investigations on the synthesis andpolymerization of functionalized 2,5-dithienylpyrrole derivatives, theelectrochemical polymerization and the properties of the parentmolecule, poly[2,5-di(2-thienyl)pyrrole], was disclosed by G. G. McLeod,M. G. B. Mahoubian-Jones, R. A. Pethuck, S. D. Watson, N. D. Truong, J.C. Galiri, and J. Francois in Polymer 27 (3), 455-8 (1986). Themolecule, 2,5-di(2-thienyl)pyrrole (structure VI), is the parentheteroaromatic monomer that forms the basis of the method of the presentinvention. Although the primary objective o McLeod et al was todetermine the solubility of the polymer resulting from2,5-di(2-thienyl)pyrrole (VI), the polymerization of2,5-di(2-thienyl)pyrrole was interesting for several additional reasons.For example, poly[2,5-di(2-thienyl)pyrrole] is readily synthesizedelectrochemically and, when anion doped, exhibits an electricconductivity analogous to polypyrrole and polythiophene. ##STR4##

However, most surprisingly and unexpectedly, and in accordance with themethod of the present invention, 2,5-di(2-thienyl)pyrrole can befunctionalized at the three-position of the pyrrole ring, and yieldconducting polymers that exhibit the high conductivity of theunsubstituted parent dithienylpyrrole (VI). As will be discussed in thedetailed description of the invention, a variety of functional groupscan be incorporated into the three-position of the pyrrole ring of2,5-di(2-thienyl)pyrrole without adversely affecting the conductivity ofthe resulting polymer.

In addition to the novel monomers used to synthesize the conductingpolymers of the present invention, the conducting polymers can befurther derivatized, after polymerization, to allow the detection andmeasurement of a specific analyte. According to the method of thepresent invention, postpolymerization derivatization andfunctionalization of the conducting polymer permits detection andmeasurement of a specific analyte by coupling the vibrational energyresulting from the functionalized polymer-analyte reaction to the phononmodes of the polymer. As used here, and throughout the specification, aphonon is a quantized, delocalized vibrational or elastic state of thepolymer lattice.

Although several references disclose the use of conducting organicpolymers in sensors, no known prior art references utilize thevibrational energy coupling of the analyte reaction to the conductingpolymer. In fact, none of the present conducting polymer-based sensorsinvolve an analyte probe molecule covalently bound to, and acting inconcert with, the polymer. In contrast, the prior art sensors are basedupon a direct interaction of an analyte, usually a gas, with thepolymer. It should be noted however that the conducting polymers used inthe present invention also can be used as an analyte sensor by directinteraction with the analyte.

The most common mode of direct interaction between the analyte and theconducting polymer is to affect the state of oxidation of the organicconducting polymer. As will be discussed more fully in the detaileddescription of the invention, the existence of bipolaron, and thereforethe conductivity of the polymer, depends upon having the polymeroxidized, with the oxidation state supported by dopant counterions.Sensors then can be developed based upon either compensating conductingfilms or chemically-doping reduced films.

For example, M. S. Wrighton et al, in European Patent No. 185,941,discloses the use of conducting organic polymers as the active speciesin a chemical sensor. The patent generally teaches using the changes inphysical properties of the conducting polymer as the active transductioninto electrical signals. Specific examples cited in the patent includedetection of oxygen gas, hydrogen gas, pH and enzyme substrateconcentrations. The Wrighton et al patent neither teaches the couplingof an analyte/probe molecule vibrational interactions to the vibrationalmanifold of the polymer nor teaches the use of such vibrational couplingas a transduction mechanism for analyte detection. In contrast, theprincipal transduction mechanism described by Wrighton et al is thedirect use of the change in polymer conductivity induced by oxidation orby reduction.

An additional mode of substrate/polymer interaction that is suitable forsensor development has been described in the prior art. It has beenshown that it is possible to utilize the change of the surfacedielectric attending the absorption of an analyte upon a polypyrrolefilm to make an alcohol sensor. In addition to novel electronictransduction mechanisms, the prior art also describes the use of asuspended gate, field effect transistor. Such electronic structures arein most ways analogous to well known structures employing inorganicsemiconductors, and they can be expected to be generically useful insensor development. In the embodiment of the invention described herein,a chemiresistor device configuration is used. It is anticipated,however, that evolutionary improvements will utilize the gatedstructures as described in the prior art.

The following references are representative of the state of the art ofelectrochemical sensors using heteroaromatic polymers:

Y. Ikariyama and W. R. Heineman, Anal. Chem. 58, 1803 (1986).

M. Josowicz and J. Janata, Anal. Chem. 58, 514 (1986).

T. N. Misra, B. Rosenberg and R. Switzer, J. Chem. Phys. 48, 2096(1968).

K. Yoshino, H. S. Nalwa, J. G. Rabe and W. F. Schmidt, Polymer Comm. 26,103 (1985).

C. Nylander, M. Armgrath and I. Lundstrom, Anal. Chem. Symp. Ser. 17(Chem Sens) 159 (1983).

H. S. White, G. P. Kittlesen and M. S. Wrighton, J. Am. Chem. Soc. 106,5317 (1984).

G. P. Kittlesen, H. S. White and M. S. Wrighton, J. Am. Chem. Soc. 106,7389 (1984).

Malmros, U.S. Pat. No. 4,444,892, disclosing a device having an analytespecific binding substance immobilized onto a semiconductive polymer toallow detection of a specific analyte.

European Patent No. 193,154, filed Feb. 24, 1986, disclosingimmunosensors comprising a polypyrrole or polythiophene film containingan occluded antigen or antibody.

M. Umana and J. Waller, Anal. Chem. 58, 2979 (1986) disclosed theocclusion, or trapping, of an enzyme, glucose oxidase, byelectropolymerizing pyrrole in the presence of the enzyme. Thepolypyrrole containing the occluded enzyme then can be used to detectglucose. The method of the present invention however differssignificantly in that according to the present invention the enzyme iscovalently bound to the conducting polymer after polymerization.

The following references are cited to further show the state of theprior art and to serve as additional background material for the methodof the present invention:

Vibrational energy transport in proteins:

A. S. Davydov, J. Theor. Biol. 38, 559 (1973).

A. S. Davydov, Physica. Scripta. 20, 387 (1979).

A. S. Davydov, studia biophysica (Berlin) 62, 1 (1977).

A. C. Scott, "Nonlinear Electrodynamics in Biological Systems," M. RossAdey and A. L. Lawrence, eds., Plenum Press, NY, 1984, p. 133.

C. F. McClare, Nature 296, 88 (1972).

A preferred synthesis of the parent molecule 2,5-di(2-thienyl)pyrrole:

H. Weinberg and J. Metselur, Syn, Comm. 14(1), 1 (1984).

The preparation of pyrrole derivatives by 1,3-dipolar cycloaddition:

(1) R. Huisgen, H. Gotthardt and H. O. Bayer, Chem. Ber. 103, 2368(1970).

(2) J. W. Lown and B. E. Landberg, Can. J. Chem. 52, 798 (1974).

SUMMARY OF THE INVENTION

In brief, the present invention is directed to analyte sensors utilizingconducting organic polymers. More particularly, the present invention isdirected to a novel class of monomers that yield conducting polymershaving substituent groups capable of functionalization. The conductingpolymers can undergo postpolymerization reactions to bond covalently toan analyte-specific probe molecule onto the polymer surface fordetection of a specific analyte and measurement of the analyteconcentration. Additionally, the conducting polymers produced accordingto the method of the present invention allow the detection andmeasurement of a specific analyte in liquid media through a newtransduction mechanism not previously observed in conducting polymers.

The analyte sensors used according to the method of the presentinvention utilize the unique electrical conducting properties ofheteroaromatic polymers to determine the presence and concentration of aspecific analyte. According to the method of the present invention, theanalyte sensors use a conducting polymer having an analyte-specificprobe molecule covalently bound to the polymer surface. The conductivityof the polymer is altered by the interaction between the probe moleculeand the analyte, and the measurable effect is detected through either adirect coupling of the vibrational interactions between theanalyte-probe molecule with the conducting polymer or through secondaryeffects produced by reaction products. If the interaction between theanalyte, probe molecule and the conducting polymer is detected through adirect linkage of the vibrational energy of the probe-analyteinteraction to the phonon-assisted bipolaron transport of the polymer,then the probe molecule must be covalently bonded to the polymer surfaceto insure vibrational coupling. Additionally, if the electricaldetection mechanism involves the chemical effect of a secondary reactionspecies, such as enzyme-substrate generated hydrogen peroxide, upon thepolymer, then direct covalent bonding between the probe molecule and thepolymer enhances detection efficiency by providing a high surfaceconcentration of the secondary reaction product.

Examples of probe molecules that can be covalently bound to theconducting polymer surface include enzymes, antigens, and ion specificbinding sites, such as crown ethers. The analyte detection mechanism inthe conducting polymer includes direct observation of molecularvibrations resulting from enzyme/substrate or antigen/antibodyreactions. As a particular example, the vibrational excitation inducedin a protein by an enzyme/substrate reaction can be transported throughthe protein in a localized waveform termed a soliton. The localizedenergy of the soliton then could be transmitted to the phonon modes ofthe conducting polymer by properly selecting the length and stiffness ofthe molecular arm covalently bridging the probe molecule and thepolymer. The conductivity of the polymer is therefore directly modulatedbecause of the dependence of the electrical properties of dopedheteroaromatic polymers upon the excitations of the internal vibrationalstates caused by the enzyme/substrate reaction.

The transducing of the probe/analyte vibrational interaction into anelectrical signal within the polymer can be assisted by a secondaryprocess. For example, the detection of the reaction product of anenzyme/substrate reaction, either through direct compensation of thedopant counterion, or, more reversibly, through the use of a counterionas a catalyst within the polymer. A specific example of this lattermechanism is the use of tetrachlororuthenate (RuCl₄ ⁻) ortetrachloroferrate (III) (FeCl₄ ⁻) ions as a dopant-catalyst for theoxidation of hydrogen peroxide. As an example, hydrogen peroxide isgenerated in the reaction of glucose oxidase with glucose in thepresence of oxygen. Therefore, by measuring the concentration ofhydrogen peroxide, the concentration of glucose in solution can beindirectly determined. The use of a dopant catalyst as an electricaltransducer in heteroaromatic polymers is disclosed in U.S. Pat. No.4,560,534 to Kung et al, and hereby incorporated by reference.

The Kung et al patent teaches using a conducting polymer, polypyrrole,doped with an anionic counterion-catalyst containing iron, ruthenium orother group VIII metals as a catalyst for hydrogen peroxidedecomposition. However, according to the method of the presentinvention, the ability to covalently couple a probe molecule to theconducting polymer surface is a significant improvement because thecovalent bond effectively enhances the transducing mechanism by insuringa high local surface concentration of the hydrogen peroxide.

In accordance with the present invention, a new class of conductingorganic polymers that are functionalized with chemically-reactivesubstituents and that maintain sufficient conductivity for use inelectrical sensors were developed. It also has been demonstrated thatprobe molecules can be covalently attached to the surface of theconducting polymer without seriously reducing the conductivity of thepolymer film. In particular, it has been demonstrated that glucoseoxidase can be covalently attached to a conducting polymer film.Moreover, it has been shown that by utilizing a covalent attachment ofthe probe molecule to the polymer surface, it is possible to design andconstruct a diagnostic device that exhibits a hydrogen peroxide doseresponse utilizing catalytic transduction.

In addition, it also has been demonstrated that the covalent bonding ofan enzyme to a conducting polymer has enabled a direct electricaltransduction of the glucose oxidase/glucose reaction. A significantfactor in the detection of glucose has been the effect of the generatedhydrogen peroxide upon the conductivity of the polymer. However,evidence exists for the operation of a direct vibrational couplingmechanism between the enzyme/substrate reaction and the conductingpolymer. In accordance with an important feature of the presentinvention, the direct vibrational coupling mechanism can occur becauseof the ability to covalently attach an enzyme, antigen or receptormolecule to the conducting polymer surface.

According to the method of the present invention, a novel class ofpolymers, demonstrating a high degree of conductivity and the capabilityof subsequent polymer surface functionalization, is generally based uponthe monomer, 2,5-di(2-thienyl)pyrrole (structure VI). Although theelectropolymerization of monomer (VI) has been reported, the prior artdoes not contain any known references pertaining to the monomersutilized to synthesize the conducting polymers of the present invention.More particularly, the conducting polymers of the present invention aresynthesized from monomers having a reactive functionality incorporatedat the three position of the pyrrole ring as shown generally instructure VII. ##STR5##

The novel feature of the monomers having the general structure VIIenabling the growth of highly conducting polymers, despite the presenceof a substituent at the three-position of the pyrrole ring, is that thecentral pyrrole ring is flanked by two thiophene rings. The resultingsteric interaction between the three-position substituent (R) with the2-and 5-position thiophene rings is decreased significantly incomparison to the corresponding terpyrrole structure that has a hydrogenatom in proximity to the three-position substituent. Thus, thethree-position substituted 2,5-di(2-thienyl)pyrrole VII can assume amore planar structure, and upon polymerization yield a film having ahigher conductivity, than its terpyrrole analog.

Analogously, and because oxidation potentials drop as oligomer sizeincreases, the following classes of molecules, depicted generally bystructures VIII, IX and X, also can serve as suitable monomers for thesynthesis of functionalized conducting polymers. Similarly, substitutedfuran monomers also can yield conducting polymers, however, theconductivity of these substituted polyfurans will be quite low due tothe decreased aromaticity of the parent rings. ##STR6##

Various members of the substituted 2,5-di(2-thienyl)pyrrole monomershaving general structure VII have been synthesized, then polymerizedelectrochemically. As will be discussed more fully hereinafter, the2,5-di(2-thienyl)pyrrole monomers of structure VII yield stable polymerfilms having conductivities significantly greater than theconductivities exhibited by the derivatized conducting polymer films ofthe prior art. It also has been found that the 2,5-di(2-thienyl)pyrrolemonomers of structure VII can be copolymerized with pyrrole, or otherlike unsubstituted parent heteroaromatics, to yield stable conductingpolymer films.

Surprisingly, in addition to synthesizing stable conducting polymersfrom the three-position substituted 2,5-di(2-thienyl)pyrrole monomers(VII), it also has been found that postpolymerization chemistry can beperformed on the three-position substituents of the pyrrole ring. Suchpostpolymerization reactions are most surprising and unexpected becausethe steric availability and the chemical environment of thethree-position substituent is modified by polymerization.

The first demonstration of polymer surface reactivity was the reactionof poly(3-acetyl-2,5-dithienylpyrrole) and phenylhydrazine to yield thecorresponding hydrazone derivative. However, this particular reactionwas difficult to monitor because the phenylhydrazine reduced thecounterion dopant, and therefore reduced the conductivity of theresulting film. Another, more useful demonstration of polymer surfacereactivity, to be discussed more fully hereinafter, was the conversionof the copolymer of3-N-trifluoroacetamidomethyl-2,5-di(2-thienyl)pyrrole (XIX) and pyrroleto the 3-aminomethyl-2,5-dithienylpyrrole copolymer by removing thetrifluoroacetyl group. Then, through any one of a variety of availablereactions, glucose oxidase was covalently attached to the free aminemoiety present on the copolymer surface.

The covalently-bound probe molecule, such as glucose oxidase, nowprovides an analyte sensor utilizing a new sensing mechanism to directlydetermine the presence and amount of an analyte, such as glucose, in aliquid medium. Furthermore, the covalent bonding of the probe moleculeto the conducting polymer offers the major advantage of monitoring theformation or decomposition of secondary reaction products, such ashydrogen peroxide. A protein probe molecule covalently bound to theconducting polymer surface allows the direct transfer of theenzyme/substrate or antigen/antibody reaction vibrational energy,possibly via soliton transport, into the phonon modes of polymer,thereby directly affecting polymer conductivity. This directtransduction of the enzyme/substrate or antigen/antibody reaction is notpossible using existing detection techniques. In fact, antigen/antibodyreactions have proved to be particularly difficult to monitorelectrically because of a lack of attendant charge transfer in thereaction.

The covalent binding of probe molecules to conducting polymer surfacesalso offers advantages in regard to secondary detection mechanisms byaffording an intimate contact between the source and the detector. Forexample, if glucose oxidase is covalently bound to the surface of theconducting polymer, the generation of hydrogen peroxide during theenzyme reaction with glucose occurs at the polymer surface. This resultsin a higher local concentration of hydrogen peroxide at the conductingpolymer surface and therefore a more efficient transduction, andsensing, mechanism. Overall, the advantages for secondary detectionmechanisms that are realized by covalently binding enzymes to theconducting polymer are analogous to the advantages offered by thesimilar covalent binding of enzymes to the active electrode inamperometric, electrochemical detectors.

Therefore, it is an object of the present invention to provide a methodof determining analyte concentrations in liquid media by utilizingorganic conducting polymers. It is also an object of the presentinvention to provide a method for determining analyte concentrations inliquid media wherein the analyte interacts with a probe molecule that iscovalently attached to the conducting polymer.

Another object of the present invention is to provide a method ofdetermining analyte con- centrations through the interaction of ananalyte with a probe molecule such that a detectable and measurableconductivity change occurs in the conducting polymer and establishes thepresence and concentration of the analyte.

Another object of the present invention is to provide a method ofdetermining analyte concentrations in liquid media from a conductivitychange in the conducting polymer caused by the reaction between thecovalently attached probe molecule and the analyte, and detected bytransferring the vibrational energy from the probe molecule-analytereaction to the conducting polymer, and the transduction of thatvibrational energy into an electrical signal.

Another object of the present invention is to provide a method ofdetermining analyte concentrations in liquid media wherein theconductivity change in the conducting polymer, caused by the reactionbetween the probe molecule and the analyte, results from secondaryprocesses of the probe molecule-analyte reaction, such as the generationand detection of hydrogen peroxide.

Another object of the present invention is to provide a conductingpolymer having substituents that can undergo postpolymerizationreactions in order to provide sites for covalent bonding of theanalyte-specific probe molecules.

Another object of the present invention is to provide a conductingpolymer that is stable to the analyte environment and that maintainspolymer conductivity over relatively long periods of time.

Another object of the present invention is to provide a conductingpolymer having substituents that can react with bridging molecules andtherefore allow probe molecules to be covalently bound to the conductingpolymer.

Another object of the present invention is to provide conductingpolymers having reactive functionalities that are protected by blockinggroups, that are unaffected by the polymerization process, and that canreact with the probe molecules or bridging molecules after removal ofthe blocking group.

Another object of the present invention is to provide monomers thatyield conducting polymers that can covalently bond to probe molecules.

Another object of the present invention is to provide monomers thatyield conducting polymers exhibiting sufficient conductivity such thatconductivity differences resulting from analyte interactions can bedetected, measured and related to analyte concentrations.

Another object of the present invention is to provide highly conductivepolymers from monomers that can undergo substituted postpolymerizationcovalent bonding to a probe molecule or to a bridging molecule.

Another object of the present invention is to provide heterocyclicaromatic monomers that yield highly conducting polymers and that aresubstituted so as to allow postpolymerization covalent bonding to aprobe molecule or to a bridging molecule.

Another object of the present invention is to provide heteroaromaticmonomers having a pyrrole or a thiophene ring substituted in thethree-position and yielding a conducting polymer exhibiting sufficientconductivity to allow detection and measurement of an analyte in liquidmedia.

Another object of the present invention is to provide heterocyclicaromatic monomers consisting of two thiophene rings, two selenophenerings, or two tellurophene rings; or a two ring heteroaromatic systemincluding a combination of a furan, a thiophene, a selenophene, and atellurophene ring; or a two ring heteroaromatic system including apyrrole ring in combination with a furan, a thiophene, selenophene, ortellurophene ring; wherein the pyrrole, if present, is substituted inthe three-position and, if pyrrole is absent, either of theheteroaromatic rings of the monomer is substituted in thethree-position.

Another object of the present invention is to provide a heterocyclicaromatic monomer including three heterocyclic aromatic compounds whereinthe two terminal heteroaromatic rings of the monomer are both furan,both thiophene, both selenophene, both tellurophene or a combination offuran, thiophene, selenophene and tellurophene; and the center ring ofthe monomer is a three position substituted thiophene, furan,selenophene, tellurophene or pyrrole ring.

Another object of the present invention is to provide heterocyclicaromatic monomers, having one five-membered heteroaromatic ringsubstituted in the three-position, wherein the ring substituent canwithstand the polymerization conditions, does not materially reduce theconductivity of the resulting conducting polymer, and can be reactedafter polymerization to covalently bond a probe molecule or bridgingmolecule to the conducting polymer.

These and other objects and advantages of the present invention willbecome apparent from the following detailed description of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the method of the present invention, organicconducting polymers are utilized as analyte sensors in diagnosticdevices to determine the presence and concentration of specific analytesin liquid media. Although organic conducting polymers have been studiedextensively, the use of conducting polymers in analyte sensors has beenimpeded by several problems, including polymer film stability, polymerconductivity, physical characteristics of the polymer, inability to testfor a specific analyte, and poor analyte detection mechanisms. As willbe described more fully hereinafter, the method of the presentinvention, surprisingly and unexpectedly, reduces or eliminates theproblems encountered in using organic conducting polymers as analytesensors.

In accordance with the present invention, a novel class of monomers thatyield highly conducting polymers has been developed. The monomers arereadily polymerized, chemically or electrochemically, to yield stablepolymers having sufficient conductivity for use as analyte sensors. Thenovel monomers, in addition to providing conducting polymers havingsuitable electrical and physical properties to act as an analyte sensor,also possess reactive substituent groups that can be functionalizedafter polymerization. In contrast to the prior art, that teachesunsubstituted pyrrole as unique because it is more easily oxidized andyields highly conducting polymers compared to ring-substituted pyrroles,it is both unexpected and surprising that the reactive substituentgroups present on the monomers used in the method of the presentinvention do not reduce the conductivity of the resulting polymer tosuch an extent that the polymer is unsuitable as an analyte sensor. Evenmore surprisingly, it has been found that the reactive substituent canundergo postpolymerization reaction and functionalization with ananalyte-specific probe molecule, such as an antigen, enzyme orion-specific binding site, without seriously affecting the electricalproperties of the polymer film.

Furthermore, it was found that the analyte-specific probe molecule canbe covalently bound to the surface of the conducting polymer. As aresult of the intimate, covalent contact between the probe molecule andthe conducting polymer surface, the vibrational interaction resultingfrom the reaction between the probe molecule and the analyte can betransferred to the surface of the conducting polymer, thereby affectingthe conductivity of the polymer. In effect, the vibrational interactionsof the probe molecule-analyte reaction are transduced into a measurableelectric signal. This electric signal then is related to the presenceand/or concentration of the specific analyte in solution. Thisvibrational energy-conductivity change analyte sensing mechanism is bothnew and unexpected in the art, and occurs because of the ability tocovalently bond a specific probe molecule to the surface of theconducting polymer either directly or indirectly through a bridgingmolecule.

In accordance with an important feature of the present invention, theproblems previously encountered in utilizing conducting organic polymersas analyte sensors in diagnostic devices are reduced or eliminated bysynthesizing conducting polymers from the novel class of monomers,generally depicted by structure XI: ##STR7## wherein A is sulfur,oxygen, selenium or tellurium, C is sulfur, oxygen, selenium ortellurium, and B is sulfur, oxygen, selenium, tellurium or N-R', whereinN is nitrogen and R' is hydrogen, an alkyl group, or a hydroxyalkylgroup. R is defined hereinafter.

In accordance with another important feature of the present invention,monomers having the general structure XI yield sufficiently conductingpolymers having reactive substituents capable of postpolymerizationreaction and functionalization. As previously discussed, such resultsare surprising and unexpected in light of the dramatic decrease inconductivity found in ring-substituted polypyrroles compared to theparent polypyrrole. However, as also previously discussed, the presenceof the aromatic thiophene, furan, selenophene and/or tellurophene ringsadjacent to the substituted pyrrole ring sufficiently decreases thesteric interaction between the ring substituent (R) and the adjacentsulfur, oxygen, selenium and/or tellurium heteroatoms.

The overall result is a class of monomers, having the general structureXI, that are essentially planar and that yield essentially planarconducting polymers having an essentially intact pi-electron system and,therefore, a relatively high conductivity. In addition, because of thesame steric and electronic effects that exist in monomers having thegeneral structure XI and because oxidation potentials drop as monomersize increases, the following monomers, illustrated by the generalstructures XII and XIII, also are expected to serve as suitable monomersfor the synthesis of highly conducting polymers that can undergopostpolymerization reaction and functionalization without adverselyaffecting the conductivity of the polymer. ##STR8##

In synthesizing monomers having the general structure XI, it was foundthat several conflicting conditions had to be satisfied. In addition tothe normal synthetic problems involved in synthesizing a three-ringmonomer, such as a pyrrole ring flanked by thiophene, furan, selenopheneand/or tellurophene rings, the placement of the reactive three-positionsubstituent (R) on the central heteroaromatic ring posed severaladditional problems. For example, the reactive three-positionsubstituent cannot be extremely electron-withdrawing orelectron-donating because large electronic effects either could alterthe oxidation potential of the monomer to such an extent thatpolymerization is precluded or, if polymerization was possible, couldadversely affect the conductivity of the polymer. Conversely, thethree-position substituent cannot be so inert, like an alkyl group, asto preclude postpolymerization reaction and functionalization of theconducting polymer.

Additionally, the reactive three-position substituent must besufficiently stable to withstand the chemical or electrochemicalpolymerization process. However, the three-position substituent must besufficiently reactive to allow the substituent to be functionalized,after polymerization, with the specific probe molecule under chemicalconditions that do not attack the conducting polymer or destroy theelectrical properties of the polymer. Finally, the reactivethree-position substituent must be sufficiently small to allow thepolymer chains to arrange themselves in sufficiently close proximity topermit charge transfer from polymer chain to polymer chain to takeplace.

In accordance with an important feature of the present invention,several monomers having the general structure XI, wherein A and C aresulfur and B is --NR'--, wherein R' is hydrogen, and having a reactivethree-position substituent meeting the above criteria and introduced onthe central ring, have been synthesized and polymerized. As seen in thefollowing examples, the synthesis of several monomers having generalstructure XI was verified by the following analytical techniques.

Infrared (IR) spectra of the monomers were obtained with a Perkin-ElmerModel 710B or 237 infrared spectrophotometer, or a Nicolet 5DBXB FT IRspectrometer unless otherwise noted; the 1602 cm⁻¹ band of polystyrenefilm was used as an external calibration standard, and absorbences arereported as cm⁻¹.

Proton magnetic resonance (¹ H NMR) spectra were obtained at 89.55 MHzusing a JEOL FX-900 spectrometer or at 60 MHz using a Varian T-60spectrometer. Spectra of the monomers were obtained using a deuteratedchloroform (CDCl₃) solution, unless otherwise noted. Chemical shifts arereported in parts per million downfield from the internal standard,tetramethylsilane (TMS).

Mass spectra (MS) were obtained using a Hewlett-Packard 5985Aspectrometer operating in either an electron impact (EI), chemicalionization (CI), or fast atom bombardment (FAB) mode.

During the synthesis of each monomer, commercial organic reagents wereused without purification, unless otherwise noted. Inorganic reagentsand reaction solvents were ACS reagent grade. Tetrahydrofuran (THF) wasHPLC grade. Brine refers to a saturated aqueous sodium chloridesolution.

Thin layer chromatography (TLC) was performed using silica gel 60F 254plates from E. Merck. Flash column chromatography was performed using E.Merck or American Scientific Products Silica Gel 60 (230-400 mesh). Allreported melting points and boiling points are uncorrected.

Elemental analyses were performed by Galbraith Laboratories, Inc. or byMiles Laboratories, Inc.

The synthetic scheme, including precursors, producing several of thesuitable monomers having the general structure XI, is found by referenceto the following examples.

EXAMPLE I N-(2-Thienylmethyl)-2-Thienylcarboxamide (XIV)

A mixture of 12.8 g (0.1 mol) of 2-thiophenecarboxylic acid and 25 mL ofthionyl chloride was stirred under reflux for 2 hours, or until hydrogenchloride and sulfur dioxide evolution stopped. The excess thionylchloride was removed under reduced pressure by azeotroping with carbontetrachloride (CCl₄), and the residue dissolved in 50 mL of diethylether. The resulting solution was added dropwise to a cold, stirredsolution containing 11.3 g (0.1 mol) of 2-thiophenemethylamine dissolvedin a mixture of 100 mL of diethyl ether and 20 mL of triethylamine.

The resulting mixture was partitioned between chloroform (CHCl₃) andwater. The organic and aqueous phases were separated. The organic phasewas washed with an 1N hydrochloric acid solution, then with a sodiumbicarbonate solution. The organic phase was dried over sodium sulfate,filtered and the CHCl₃ evaporated to yield 21.83 g (98%) of a yellowsolid. TLC (silica gel); 60:10:1 [CHCl₃ :methanol (CH₃ OH):concentratedammonium hydroxide (NH₄ OH)] showed one product.

A portion of the product was recrystallized from CHCl₃ /diethyl ether toyield a white solid having a melting point (mp) of 115°-117° C.

Analysis: Calc'd for C₁₀ H₉ NOS₂ : C,53.78; H, 4.06; N, 6.27 Found:C,53.68; H, 3.82; N, 6.48

¹ H NMR (60 MHz, CDCl₃)δ: 4.7 (d, 2H, --NH--CH₂ --); 6.8-7.8 (m, 6H)

IR (CHCl₃)cm⁻¹ : 3450, 1660, 1550 ##STR9##

EXAMPLE II N-(2-Thienylmethyl)-2-Thienyliminochloride (XV)

A cold (0° C.) solution including 27 g of phosgene in 140 mL of CHCl₃was treated with 1.5 mL of N,N-dimethylformamide (DMF), then a solutioncontaining 15.44 g of compound XIV (69 mmol) in 100 mL of CHCl₃ wasadded dropwise over a 0.5 hour period. The resulting mixture was stirredfor 1 hour at 0° C., then was allowed to warm to ambient temperatureovernight (approximately 21 hours).

An aliquot of the reaction mixture was withdrawn via syringe and thesolvents removed in vacuo. The resulting oil was azeotropicallydistilled with CCl₄. The product residue gave the following spectraldata:

IR(CDCl₃)cm⁻¹ : 1650

¹ H NMR(60 MHz, CDCl₃)δ: 5.08 (s, 2H); 7.0-7.8 (m, 6H)

TLC (SiO₂, 9:1 toluene:dioxane): R_(f) =0.1

The solvents were evaporated at 40° C. in vacuo from the bulk of thereaction mixture to yield a dark red oil. The oil was triturated withdiethyl ether and the combined filtrates filtered through CELITE(Manville Products Corp., Denver, Colo. 80217). The diethyl ether thenwas removed in vacuo. The compound XV was obtained by evaporativedistillation at 116°-140° (0.1 mm), yielding 14.4 of XV as a lightyellow oil (86%).

EXAMPLE III 3-Cyano-3-[H]-4,5-Dihydro-2,5-Dithienylpyrrole (XVI)

To a cold (-30° C.) stirred solution, protected by an inert atmosphere,containing 15.66 g of (65 mmol) compound XV and 32 g (330 mmol) ofacrylonitrile in 65 mL of dry DMF was added 8.5 g of1,5-diazabicyclo[4.3.0]non-5-ene (DBN, 66 mmol) dropwise. The resultingmixture was stirred at -30° C. for 1.5 hours. The excess acrylonitrilethen was evaporated at 50° C., and 17 mm pressure. DMF was removed byevaporation at 50° C., and 0.1 mm pressure. The residue was partitionedbetween CHCl₃ and water, filtered through CELITE to removepolyacrylonitrile, and the organic and aqueous layers separated. TheCHCl₃ layer was dried over magnesium sulfate, filtered, and evaporated.The resulting oil was dissolved in toluene and chromatographed on 200 gof SiO₂ -60 eluted with a 1% dioxane-toluene solvent mixture. Fifteenmilliliter-sized fractions were collected.

Fractions numbered 16 to 40 contained one regioisomer (analytical TLC,SiO₂, 1% dioxanetoluene, R_(f) =0.5 visualization with ceric ammoniumnitrate spray reagent). Fractions numbered 16 to 40 were combined andconcentrated to yield 6.6 g of an oil that eventually solidified (39%yield).

The following analytical data was obtained in regard to the structure ofthis regioisomer:

¹ H NMR (60 MHz, CDCl₃)δ: 3.0-3.8 (m, 3H, pyrrole C₃ -H, C₄ -H₂); 5.68(dd, pyrrole C₅ -H); 7.0-7.6 (m, 6H, thienyl C--H).

An analytical sample of this regioisomer (mp 120.5°-122.5° C.) wasprepared from a CHCl₃ -hexane solvent mixture.

Analysis: Calc'd. for C₁₃ H₁₀ N₂ S₂ : C, 60.43; H, 3.90; N, 10.84;Found: C, 61.15; H, 4.19; N, 10.76

Mass Spectrum (EI) m/e=258.3 (M⁺, 45.9%) 259.0 (M+1, 9.4%)

Fractions numbered 41 through 49 were combined and concentrated to yield0.29 g (2% yield) of an oil containing a mixture of the regioisomerfound in fractions 15-40 and a second regioisomer (R_(f) =0.4).Fractions numbered 50 through 79, after combining and concentratingyielded 4.9 g of an oil containing only the regioisomer of R_(f) =0.4and having the following spectral characteristics:

¹ H NMR (60 MHz, CDCl₃)δ: 3.2-3.8 (m, 3H, pyrrole C₃ -H, C₄ -H₂) 5.8 (d,J=8Hz, pyrrole C₅ -H); 7.0-7.6 (m, 6H, thienyl C--H)

An analytical sample of the second regioisomer (mp 105°-106°) wasprepared by recrystallization from methylene chloride (CH₂ Cl₂).

Analysis: Calc'd. for C₁₃ H₁₀ N₂ S₂ : C, 60.43; H, 3.90; N, 10.84 Found:C, 55.54; H, 3.78; N, 9.99 (12% CH₂ Cl₂)

Mass Spectrum (EI) m/e=258 (M⁺, 30%).

In all later syntheses, the crude reaction mixture, containing bothdiastereomers, was used without further purification. ##STR10##

EXAMPLE IV 3-Cyano-2,5-Dithienylpyrrole (XVII)

A solution containing 13.05 g of crude3-cyano-3-[H]-4,5-dihydro-2,5-dithienylpyrrole (XVI) and 2.9 g of 10%Pd/C in 135 mL of diphenyl ether was heated at 195° C. for 5 hours undera purging stream of carbon dioxide (CO₂). The reaction mixture then wascooled and subsequently filtered through CELITE. The filter cake waswashed with CHCl₃ and the combined filtrates were evaporated in vacuofirst at 12 mm pressure, then at 1.2 mm pressure, to remove the reactionsolvents. The residue (10.14 g) was recrystallized from anacetone-toluene mixture to yield 3.14 g of the product XVII (24.2%yield) as a greyish-yellow solid (mp 202°-203.5° C.).

Analysis: Calc'd. for C₁₃ H₈ N₂ S₂ : C, 60.91; H, 3.15; N, 10.93 Found:C, 61.16; H, 3.26; N, 11.06

The mother liquor was concentrated to give 8.08 g of a viscous oil thatwas flash chromatographed on 250 g of SiO₂ eluted with a 19:1toluene-THF solvent mixture. Fractions numbered 36 through 64,containing the reaction product, were combined, then concentrated, toyield 2.5 g of a yellow solid that was recrystallized fromacetone-toluene to give an additional 1.26 g of compound XVII (9.7%yield). An additional 489 mg of the compound XVII was similarly obtainedfrom the mother liquor (3.8% yield) by repeating the above procedure.The total isolated yield of compound XVII was therefore 37.7%.

IR (KBr) cm⁻¹ : 3210, 3160, 2210

¹ H NMR (90 MHz, DMSO-d₆)δ: 6.8 (d, J=2 Hz, 1H); 7.1 (q, J=4 Hz, 2H);7.46 (d, J=4 Hz, 2H); 7.7 (d, J=4 Hz, 2H).

¹³ C NMR (22.5 MHz, DMSO-d₆)δ: 90.1, 109.8, 116.7, 124.0, 125.0, 126.0,126.7, 127.8, 128.2, 131.2, 133.2, 133.7

Mass Spectrum (EI) m/e: 256.3 (M⁺, 100.0%) 257.1 (M+1, 22.4%).

EXAMPLE V 3-Aminomethyl-2,5-Dithienylpyrrole (XVIII)

To a stirred solution of 0.8 g (3 mmol) of 3-cyano-2,5-dithienylpyrrole(XVII) in 20 mL of dry THF was added 8 mL of a 1M solution ofborane-tetrahydrofuran complex in THF. After the initial exothermicreaction subsided, the mixture was heated to reflux under an inert(argon gas) atmosphere overnight. The solvents then were evaporated invacuo and the residue partitioned between CHCl₃ and a 3N hydrochloricacid (HCl) solution. The CHCl₃ layer then was extracted three times with10 mL portions of 3N HCl dried over sodium sulfate, filtered, andconcentrated in vacuo to give 410 mg of unreacted3-cyanodithienylpyrrole (XVII).

The combined aqueous acidic solutions were made basic with a sodiumhydroxide (NaOH) solution and then were extracted with CHCl₃. Theresulting CHCl₃ solution was dried over magnesium sulfate, filtered, andconcentrated to yield 360 mg of a solid. Recrystallization of the solidfrom hot toluene gave 100 mg of compound XVIII (mp 154°-155° C.).

Analysis: Calc'd. for C₁₃ H₁₂ N₂ S₂ : C, 59.96; H, 4.65, N, 10.76 Found:C, 59.99; H, 4.61; N, 10.5

¹ H NMR (60 MHz, CDCl₃)δ: 3.8 (s, 2H); 1.95 (m, 3H); 6.5 (s, 1H);7.1-7.3 (m, 6H)

Mass Spectrum (EI) m/e: 260.1 (M⁺, 100%), 244.1 (M⁺ --NH₂, 100%).

The mother liquor was concentrated and chromatographed by preparativeTLC plates (SiO₂ -60, 20 cm×20 cm×1000u) eluted successively with CHCl₃and a 60:5:1 CHCl₃ -CH₃ OH-concentrated NH₄ OH solvent mixture. Thechromatographic band containing the product XVIII was excised andextracted with hot ethanol. The ethanol solution was filtered, thenconcentrated in vacuo to yield an additional 100 mg of compound XVIII.The combined yield of compound XVIII was 460 mg (59% yield). ##STR11##

EXAMPLE VI 3-N-Trifluoroacetamidomethyl-2,5-dithienylpyrrole (XIX)

A mixture containing 1.64 g (6.31 mmol) of3-aminomethyl-2,5-dithienylpyrrole (XVIII) in 75 mL of CHCl₃ was cooledto 0° C. and treated successively with 7.5 mL of ethyl trifluoroacetateand 1.0 mL of diisopropylethylamine. The mixture was allowed to warm toambient temperature and produced a homogenous solution. After two hoursat ambient temperature, an additional 2 mL of ethyl trifluoroacetate and0.5 mL of diisopropylethylamine was added to the mixture. The mixturewas stirred overnight, then heated to reflux for ten minutes. Aftercooling, the mixture was concentrated in vacuo to yield an oil. Theresidual oil was flash chromatographed on 250 g of SiO₂ -60 (230-400mesh) eluted with CHCl₃. Eighteen milliliter-sized fractions werecollected, and fractions numbered 18 through 42 were combined andconcentrated to yield 2.11 g of a brown foam. Recrystallization of thebrown foam, with seeding, from a 2:1 toluene-hexane mixture, gave 1.51 g(70% yield) of a pink-beige powder (mp 107°-109° C.).

Analysis: Calc'd. for C₁₅ H₁₁ F₃ N₂ OS₂ : C, 50.55; H, 3.11; N, 7.86Found: C, 50.47; H, 3.12; N, 7.54

¹ H NMR (90 MHz, CDCl₃)δ: 4.55 (d, J=5Hz, 2H); 6.40 (m, NH); 6.42 (d,pyrrole C₂ -H); 7.0-7.3 (m, 6H); 8.4 (m, NH)

¹³ C NMR (22.5 MHz, CDCl₃)δ: 36.7, 108.7, 117.1, 121.9, 123.6, 124.4,124.8, 125.0, 127.4, 127.8, 133.0, 134.8

IR (KBr) cm⁻¹ : 3300, 3100, 1700, 1550, 1210, 1190, 1170.

EXAMPLE VII 3-(2-Hydroxyethyl)-2,5-Dithienylpyrrole (XX)

A solution containing 2.31 g (10 mmol) of 2,5-di(2-thienyl)pyrrole (VI)in 100 mL of dry diethyl ether, maintained at 0° C., was treated with6.3 mL of a 1.6M solution of n-butylmagnesium bromide (10 mmol) indiethyl ether. The resulting slurry was stirred for 0.5 hour at 0° C.and then was treated with 3 g of ethylene oxide. The mixture was stirredfor 1 hour at 0° C. Dry THF (100 mL) was added to produce a homogeneoussolution and the solution was stirred for 1 hour at 0° C. The solutionwas allowed to reach ambient temperature over 1 hour period, then thereaction was quenched by adding 1 mL of a saturated ammonium chloride(NH₄ Cl) solution. The resulting mixture was filtered, and the solventsevaporated in vacuo in the presence of 25 g of SiO₂ -60. The solidabsorbed on the SiO₂ -60 was chromatographed on a 200 g column of SiO₂-60 using a 1% dioxane-toluene solvent mixture. Fractions of 15 mL involume were taken. Fractions numbered 111 through 215 were combined andconcentrated to yield 1.36 g of an oil, containing of a mixture of thedesired compound XX and approximately 17% of a contaminant identified asN-(2-hydroxyethyl)-2,5-di(2-thienyl)pyrrole, as determined by ¹ H NMR.The mixture was used without further purification.

¹ H NMR (60 MHz, CDCl₃)δ: 3.0 (t, J=6Hz, pyrrole-C₃ -CH₂ -CH₂ -OH); 3.4(m, N-CH₂ -CH₂ -OH); 3.9 (t, J=6Hz, pyrrole-C₃ -CH₂); 4.4 (t, J=6Hz,N-CH₂ -); 6.4 (d, J=2Hz); 6.9-7.4 (m, 6H); 8.93 (m, NH)

Mass Spectrum (EI) m/e 275.1 (M⁺, 56.3%) ##STR12##

EXAMPLE VIII 3-(2-Phthalimidoethyl)-2,5-dithienylpyrrole (XXI)

A solution containing 0.75 g of methanesulfonyl chloride in 25 mL of CH₂Cl₂ was added to a stirred solution containing 0.8 g (3.3 mmol) of3-(2-hydroxyethyl)-2,5-dithienylpyrrole (XX) and 3 mL of triethylaminein 25 mL of methylene chloride (CH₂ Cl₂) maintained at 0° C. under aninert argon gas atmosphere. The mixture was stirred for 2 hours at 0°C., followed by treatment with a solution containing 8 g (40 mmol) ofpotassium phthalimide in 50 mL of DMF. The resulting mixture was heatedat 40° C. overnight. After cooling to room temperature, the mixture wasfiltered, and the solvents of the filtrate evaporated in vacuo in thepresence of with 20 g of SiO₂ -60. The solid absorbed on the SiO₂ waschromatographed on a 100 g column of SiO₂ -60 that was equilibrated andeluted with a 1% dioxane-toluene solvent mixture. Fractions havingvolumes of 20 mL were collected, and fractions numbered 10 through 25,containing the product (XXI), were combined, then concentrated to yield860 mg of an oil (64% yield). An analytical sample of compound XXI (mp174°-175° C.) was recrystallized from diethyl ether.

Analysis: Calc'd. for C₂₂ H₁₆ N₂ O₂ S₂ : C, 65.32; H, 3.99; N, 6.93Found: C, 65.06; H, 4.03; N, 6.71

¹ H NMR (60 MHz, CDCl₃)δ: 3.0 (t, J=7Hz, 2H); 3.9 (t, J=7Hz, 2H); 6.4(d, J=3Hz, 1H); 6.9-7.3 (m, 6H); 7.7 (m, 4H); 8.5 (m, NH)

IR (CHCl₃)cm⁻¹ : 3450, 3010, 1780, 1720, 1405, 1370

Mass Spectrum (EI) m/e: 404.1 (M⁺, 39.4%) 405 (M⁺¹, 11.2%)

EXAMPLE IX 3-(2-Aminoethyl)-2,5-dithienylpyrrole (XXII)

A solution of 800 mg of 3-(2-phthalimidoethyl)-2,5-dithienylpyrrole(XXI) and 100 mg. of 95% hydrazine (3 mmol) in 25 mL of ethanol washeated to reflux for 3 hours. The mixture then was cooled and dilutedwith 25 mL of 1N HCl. The ethanol was removed in vacuo, and theresulting aqueous solution was filtered. The filtrate was made alkalinewith sodium hydroxide (NaOH) and extracted with CHCl₃. The organic CHCl₃layer was dried over magnesium sulfate, filtered, and the CHCl₃evaporated to give 0.48 g of a yellow oil. The product was isolated bypreparative SiO₂ TLC (20 cm×20 cm×1000u) using a 120:10:1 CHCl₃ -CH₃OH-conc.NH₄ OH solvent mixture. Three hundred mg of a solid wasobtained. The solid was recrystallized from a toluene-hexane solventmixture and dried at 55° C. at 0.1 mm pressure gave 200 mg of compoundXXII (24% yield, mp 136°-138° C.).

Analysis: Calc'd. for C₁₄ H₁₄ N₂ S₂ : C,61.28; H, 5.14; N, 10.21 Found:C,61.20; H, 5.15; N, 9.81

¹ H NMR (60 MHz, CDCl₃)δ: 2.0 (m, NH₂); 2.8 (m, 4H); 6.3 (s, 1H);6.8-7.4 (m, 6H)

IR (CHCl₃)cm⁻¹ : 3430, 2920, 1590, 1520, 1430, 1270

Mass Spectrum (EI) m/e: 274.0 (M⁺, 35%) ##STR13##

EXAMPLE X 3-(2-Trifluoroacetamidoethyl)-2,5-dithienylpyrrole (XXIII)

A solution containing 100 mg (0.36 mmol) of3-(2-aminoethyl)-2,5-dithienylpyrrole (XXII), 2 mL of ethyltrifluoroacetate, and 5 mL of CHCl₃ was allowed to stand at ambienttemperature overnight. The CHCl₃ solvent was removed in vacuo, and theproduct XXIII isolated by preparative SiO₂ -TLV plates (20 ×20 ×1000u)eluted with a 9:1 toluene-dioxane solvent mixture. Sixty mg of compoundXXIII was obtained (45% yield).

¹ H NMR (60 MHz, CDCl₃)δ: 2.87 (t, 2H); 3.5 (t, 2H); 6.26 (d, 1H,pyrrole C₃ -H); 6.8 (m, 1H, NH); 7.0 (m, 6H); 8.4 (m, NH)

Mass Spectrum (EI) m/e: 370.2 (M⁺, 75.9%).

EXAMPLE XI 3-Acetyl-2,5-Dithienylpyrrole (XXIV)

A mixture containing 13.25 g (53 mmol) of 1,4-dithienyl-1,4-butanedione,38.9 g (0.53 mol) of ammonium acetate, 53 mL of acetic anhydride, and212 mL of acetic acid was stirred under reflux in an inert argon gasatmosphere for 12 hours. The solvents then were removed in vacuo and theresidue partitioned between CHCl₃ and water. The organic and aqueouslayers were separated and the organic solvents evaporated in vacuo toyield a solid. Analytical TLC showed that the solid was a mixture ofstarting materials and several reaction products. The crude solid wasdissolved in a mixture containing 38.9 g of ammonium acetate, 106 mL ofacetic anhydride, and 212 mL of acetic acid and heated to refluxovernight. The resulting mixture was cooled and concentrated by removingthe solvents in vacuo. The residue was partitioned between diethyl etherand water. A solid precipitated from the aqueous layers, and the solidwas filtered, dried, and found to be unreacted starting materialdiketone (8.6 g). The filtrate and the diethyl ether layer were found tocontain a mixture of three components. The diethyl ether layer was driedover magnesium sulfate, filtered, and the ether removed in vacuo toyield a dark oil. Crystallization of the oil from toluene yielded 1 g ofa solid identified as 3-acetyl-2,5-dithienylpyrrole (XXIV), mp 181°-183°C.

Analysis: Calc'd. for C₁₄ H₁₁ NOS₂ : C,61.51;H,4.06; N,5.12 Found:C,61.43; H,4.31; N,5.02

¹ H NMR (60 MHz, CDCl₃)δ: 2.39 (s, 3H); 6.78 (d, J=3Hz, 1H, pyrrole C₃-H); 6.95-7.4 (m, 5H); 7.55 (dd, J=3Hz, 1H); 9.07 (m, NH)

IR (CDCl₃)cm⁻¹ : 3400, 3200, 1668

Mass Spectrum (EI) m/e: 273.0 (M⁺, 82.3%). ##STR14##

EXAMPLE XII 3-Carboxyethyl-2,5-Dithienylpyrrole (XXV)

A mixture containing 22.42 g (93 mmol) ofN-(2-thienylmethyl)-2-thienyliminochloride (XV), 10.8 g (110 mmol) ofethyl propiolate, 12.5 g of freshly distilled DBN, and 150 mL of dry DMFwas stirred at 0° C. for 0.5 hour, then allowed to warm to ambienttemperature over a 3.5 hour period. The reaction solvents were removedin vacuo at 50° C. and the residue dissolved in CH₂ Cl₂. The organicmixture was washed successively three times with 5% aqueous HCl, water,and 5% aqueous NaHCO₃. After drying over magnesium sulfate, filteringand removing the organic solvents in vacuo, the resulting oil wasdistilled. The fraction distilling at 190°-220° C. was chromatographedon a 100 g column of SiO₂ -60 eluted with toluene. Fractions numbered 31through 95, 15 ml in each volume, contained the desired compound (XXV)(R_(f) =0.27). The fractions were combined, then concentrated to yield2.5 g of an oil. Crystallization of the oil from hexane gave 600 mg ofcompound XXV (2% yield, mp 103°-104° C.).

Analysis: Calc'd. for C₁₅ H₁₃ NO₂ S₂ : C, 59.38; H, 4.32; N, 4.62 Found:C, 59.13; H, 4.46; N, 4.51

¹ H NMR (60 MHz, CDCl₃)δ: 1.45 (t, J=7Hz, 3H); 4.55 (q, J=7Hz, 2H); 6.88(d, J=3Hz, 1H); 7.0-7.6 (m, 5H); 7.65 (dd, J=3Hz, 1Hz, 1H)

IR (KBr) cm⁻¹ : 3430, 2970, 1700, 1600, 1450, 1260, 1115.

EXAMPLE XIII3-[(N-3-Carbomethoxypropionyl)aminomethyl]-2,5-Dithienylpyrrole (XXVI)

To a solution of 400 mg (1.54 mmol) of3-aminomethyl-2,5-dithienylpyrrole (XVIII) and 378 ul (3.07 mmol) ofdiisopropylethylamine in 15 ml of anhydrous methylene chloride was added3-carbomethoxypropionyl chloride (1.6 ml, 9.23 mmol). After stirring for5 minutes at room temperature, the reaction mixture was quenched by theaddition of 1 ml of water. The aqueous layer was extracted three timeswith 5 ml portions of chloroform and the combined organic layers weredried over anhydrous magnesium sulfate, filtered, and concentrated invacuo. The crude reaction product was chromatographed on 100 g of silicagel. Elution with chloroform:methanol, 95:5, gave 456 mg (39%) of theamide (XXVI) as a greenish solid having a mp of 48°-49° C., and R_(f):0.55 (chloroform:methanol, 98:2).

IR (KBr)cm⁻¹ : 3400, 1735, 1650, 1530, 1440, 1220, 1170, 860, 695

¹ H NMR (CDCl₃, 60MH_(z))δ: 6.90 to 7.36 (m, 7H); 6.42 (M, 1H, pyrroleC4-H); 5.70 to 6.00 (brs, 1H, -NH-); 4.33 to 4.63 (M, 2H, CH₂ -N);3.67(s, 3H, -CO₂ CH₃); 2.60 (t, 2H, J=5Hz -CH₂ -CO-); 2.51 (t, 2H,J=5H_(z), -CH₂ CO)

Mass spectrum (EI) m/e: 374.2 (M⁺, 100%). ##STR15##

EXAMPLE XIV 3-[(N-3-Carboxypropionyl)-aminomethyl]-2,5-Dithienylpyrrole(XXVII)

A mixture of compound XXVI (200 mg, 0.53 mmol), 0.6N aqueous sodiumhydroxide (1.34 ml, 0.80 mmol) and 4 ml of methyl alcohol was stirred atroom temperature for 16 hours. The reaction mixture was poured onto 15ml of ice water and was washed with diethyl ether. The aqueous layer wasacidified with 1N aqueous hydrochloric acid to pH 4.0, then saturatedwith sodium chloride. The saturated solution then was extracted fourtimes with 5 ml portions of ethyl acetate. The combined organic extractswere washed with the 5 ml portions of water, dried over anhydrousmagnesium sulfate, then filtered. Evaporation of solvent in vacuo gave acrude acid that was then purified on 20 g of silica gel. Elution withethyl acetate:acetic acid, 95:5, provided 121 mg (63%) of acid (XXVII),having R_(f) :0.28 (ethyl acetate:acetic acid, 95:5).

IR (KBr)cm⁻¹ : 2700-3400, 1715, 1640, 1535, 1410, 1210, 1175, 1020,1000, 840, 820, 690.

¹ H NMR (CDCl₃, 60 MH_(z))δ: 9.90 to 10.36 (brs, 1H, CO₂ H); 9.63 to9.90 (brs, 1H, -NH); 6.83 to 7.33 (m, 7H); 6.33 (d, 1H, J=3H_(z),pyrrole C-4 protons); 4.43 (d, 2H, J=2Hz, CH₂ -NH); 2.26 to 2.70 (m, 4H,-CO-CH₂ -CH₂ CO-)

Mass spectrum (EI) m/e: 360.1 (M⁺, 55.9%), 260.1 (M⁺ -CO(CH₂)₂ CO₂ H,70%), 244.1 (M⁺ -NH₂ -CO(CH₂)₂ CO₂ H, 100%).

From the above examples, it is seen that a number of differentsubstituent groups (R) can be introduced into the three-position of thecentral heteroaromatic ring of the monomer. In particular, the examplesshow that the three-position substituent on monomers having the generalstructure VII can be carboxyethyl (XXV), acetyl (XXIV), cyano (XVII),aminomethyl (XVIII), aminoethyl (XXII), N-trifluoroacetamidomethyl (XIX)and N-trifluoracetamidoethyl (XXIII). However, in accordance with themethod of the present invention, other substituents can be introducedinto the three-position of the molecule. It also is possible to placesubstituents on the 4-position of the molecule, if such substituents donot materially affect the polymerizability of the monomer and theconductivity of the polymer.

To achieve full advantage of the present invention, it is necessary tohave the exposed amino functionality of the monomer3-aminomethyl-2,5-dithienylpyrrole (XVIII) available on the surface ofthe conducting polymer. This amino functionality is ideally suited toeither covalently bond directly to an enzyme, antigen or other specificbinding molecule, or covalently bond to a spacer or bridging moleculebefore subsequent bonding to the enzyme, antigen, or other specificbinding molecule. The introduction of the exposed amino moiety on thesurface of the conducting polymer can be effected either directly bypolymerization of the monomer XVIII, or by polymerization of a monomerwherein the amino functionality is protectively blocked, followed byremoval of the blocking group to expose the amino moiety on the polymersurface. The preferred monomer that includes the blocked aminofunctionality is 3-N-trifluoroacetamidomethyl-2,5-dithienylpyrrole(XIX). The monomer XIX is preferred not only because of its ease ofsynthesis and facile polymerization, but also because the monomer XIXaffords polymers exhibiting unusual and surprising conductivity, even incomparison to polymers grown from monomer XVIII. In addition,protectively blocking the amino group enables polymer growth to beconducted in electrolyte solutions containing ions such astetrachlororuthenate (RuCl₄ ⁻). It is not possible to grow a polymerfrom the free amine monomer (XVIII) in the presence oftetrachlororuthenate ions because of adduct formation.

Other amino protecting groups providing better stability in the event ofextreme acid or base conditions also can be used according to the methodof the present invention. The protecting groups listed in Table 1 can becleaved from the conducting polymer under conditions that are milderthan the conditions required to remove the N-trifluoroacetyl group.Therefore, if necessary, the conducting polymer is protected from therelatively harsh conditions required to remove the N-trifluoroacetylgroup.

                                      TABLE 1                                     __________________________________________________________________________    AMINE N-PROTECTING GROUPS                                                     PROTECTING              DEPROTECTION                                          GROUP     STRUCTURE     CONDITIONS                                            __________________________________________________________________________    N-Dithiasuccinoyl                                                                        ##STR16##    Thioethanol/Triethanolamine 25° C., 5                                  min.                                                  Vinyl Carbamate                                                                          ##STR17##    Anhydrous hydrogen chloride/dioxane, 25°                               C.; or hydrogen bromide/acetic acid                   t-Butyl Carbamate                                                                        ##STR18##    3M Hydrochloric acid/ethyl acetate 30 min.;                                   trifluoroacetic acid 0° C., 5 min;                                     iodotrimethylsilane, CHCl.sub.3 or acetonitrile                               25° C., 6 min.                                 o-Nitrothiophenol                                                                        ##STR19##    22° C., 1 hr.; 2-mercaptopyridine,                                     CH.sub.2 Cl.sub.2, 1 min.; acetic acid, aqueous                               alcohol or hydrochloric acid, alcohol, 1 hr.          N-Trifluoracetamide                                                                      ##STR20##    Sodium methoxide/ methanol 25° C., 16          __________________________________________________________________________                            hrs.                                              

Similarly, in accordance with the method of the present invention,monomers having a longer N-functionalized amido spacer, or bridging arm,than the 3-aminomethyl dithienylpyrrole (XVIII) can be synthesized andpolymerized. Conducting polymer films synthesized from the monomershaving longer spacer arms, like monomers XXVI and XXVII, allow for themore efficient covalent attachment of the probe molecule, like an enzymeor an antigen, to the conducting polymer. However, the additional stericbulk of these longer spacer arms may adversely affect the growth of thepolymeric film and the conductivity properties of the polymeric film.Examples of other longer spacer arm derivatives of 3-aminomethyldithienylpyrrole are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        FUNCTIONALIZED SPACER ARM DERIVATIVES                                         OF 3-AMINOMETHYL DITHIENYLPYRROLE                                             ______________________________________                                         ##STR21##                                                                     ##STR22##                                                                     ##STR23##                                                                     ##STR24##                                                                    ______________________________________                                    

The method of the present invention allows antigen or enzymeimmobilization on other dithienylpyrrole derivatives in addition to the3-aminomethyl dithienylpyrrole derivative (XVIII). For example, the2-hydroxyethyl derivative (XX) can be modified to provide protected andpolymerizable thiol, carboxaldehyde, or carboxylate monomers.Additionally, unprotected hydroxyethyl dithienylpyrrole (XX) yields aconducting film capable of surface modification by eitheraminopropylsilation or oxidative chemical treatment prior to probemolecule attachment.

In accordance with the method of the present invention, conductingpolymers can be synthesized from monomers having a pyrrole as thecentral heteroaromatic ring, and also from terthienyl monomers(structure XI, wherein A, B and C are sulfur) that are substituted atthe three-position of the central thiophene ring. In general, theterthienyl monomers will yield conducting polymers having increasedchemical stability making them especially useful in the preparation ofconducting polymers that subsequently will be subjected to stronglyalkaline conditions. Similarly, other heteroaromatic monomers, having athree-position substituted selenophene, tellurophene or furan ring asthe central heteroaromatic ring of structure XI, can be used tosynthesize conducting polymers.

Furthermore, in addition to the three-position substituents placed onthe heteroaromatic monomer of general structure VII and described in theprevious Examples, other reactive substituents that can be placed at thethree-position of the heteroaromatic ring and that can be covalentlybound to a probe molecule without adversely affecting the conductivityof the polymer include:

    __________________________________________________________________________     ##STR25##                                VII                                 Derivative of VII  R-Group          Reactive With                             __________________________________________________________________________    3-(2-methyldithioethyl)                                                                          CH.sub.2 CH.sub.2SSCH.sub.3                                                                    HS-(Protein,                                                                  Fab, Enzyme)                              3-(N-imidazocarbonyl)amidomethyl                                                                  ##STR26##       NH.sub.2 -Protein                         3-(4-nitrophenylcarbamoyl)amidomethyl                                                             ##STR27##       NH.sub.2 -Protein                         3-(formylmethyl)   CH.sub.2CHO      NH.sub.2 -Protein                                                             (with or without                                                              Glutaraldehyde)                           3-(carboxymethyl)  CH.sub.2CO.sub.2 H                                                                             NH.sub.2 -Protein                                                             water soluble                                                                 carbodiimide                              __________________________________________________________________________

In accordance with another important feature of the present invention,the 2,5-di(2-thienyl)pyrrole monomers previously described can bechemically or electrochemically polymerized to yield conducting organicpolymers. In the preferred synthetic method, the conducting polymer issynthesized electrochemically in order to obtain the polymer directly inits oxidized state. During the electrochemical synthesis, the conductingpolymer incorporates the anion of the supporting electrolyte into thepolymer structure, usually in a ratio of approximately four monomerunits per anion, thereby producing the polymer in its oxidized state.

Electrochemical techniques can be used to drive the anion from thepolymer to yield an insulating, non-oxidized polymer; and similarlyelectrochemical techniques can be used to oxidize, or introduce theanion, to an insulating, non-oxidized polymer. The ability to reversiblyoxidize conducting polymers is extremely important and is directlyconnected to the stability of the conducting polymer. It should also benoted that electrochemical synthesis of the conducting polymer ispreferred because the thickness of the conducting polymeric film can beeasily and precisely controlled by monitoring the electrolysis time. Inregard to stability, it is preferred that the conducting organic polymeris stable in the presence of water and under prolonged water exposurebecause a majority of the analytes of interest exist in aqueous media.

The conducting polymer films made according to the method of the presentinvention were synthesized electrochemically. The usual conditions forpolymer synthesis involve growth of the conducting polymer under anargon gas atmosphere at an anodic potential of 0.8 V with respect tosilver/silver ion (Ag/Ag⁺) reference electrode in an 5×10⁻³ M monomersolution in dry acetonitrile (CH₃ CN) solvent that has been purged withargon gas (Ar). The dopant counterion is present at a concentration of0.01M.

In general, the electropolymerization process requires a workingelectrode and an electrolytic medium that includes the monomer, anorganic solvent and a supporting electrolyte. The conducting polymer, inits doped and oxidized state, is grown on the working electrode. Theworking electrode can be a metal, such as gold, platinum, aluminum,rhodium, titanium, tantalum, nickel or stainless 314; a metal oxide suchas tin oxide, titanium oxide, or indium tantalum oxide; semiconductingsubstances such as silicon, germanium, gallium arsenide, cadmium sulfideor cadmium selenide; carbonaceous substances, such as graphite or glassycarbon; or other suitable electrode materials.

The choice of solvent for the electrolyte medium directly affects thephysical, morphological and electrical characteristics of the conductingpolymer. If an organic solvent is employed, it is preferred that thesolvent be both aprotic and a poor nucleophile in order to ensure thatthe solvent itself does not become directly involved in the electrolyticreactions. Typical solvents that have been used in the electrochemicalsynthesis of the conducting polymers of the present invention includeacetonitrile, tetrahydrofuran, methylene chloride, benzonitrile,dimethyl sulfoxide, ethanol, propylene carbonate,hexamethylphosphoramide, butanone and nitromethane.

The supporting electrolyte is a critical component in the electrolytemedium because the supporting electrolyte is directly incorporated intothe conducting polymer as a dopant that compensates the charge carriersof the organic polymer. If the conducting polymer is grownelectrochemically, the inclusion of the anionic dopant within theconducting polymer film is a direct and integral part of the polymergrowth process. The conducting polymer film, as grown, is fully doped,with the anionic electrolyte included within the conducting polymer atamounts ranging from about 10 to about 35 atomic percent. The conductingpolymer film, once grown, can be reversibly reduced and reoxidized withthe release and reinclusion of the dopant counterions by the filmattending the reduction and oxidation processes.

Electrochemical synthesis of the conducting polymer permits theselection of a particular counterion from a wide variety ofelectrolytes. The choice of the supporting electrolyte is importantbecause the electrolyte will affect both the electroactivity and thestructural properties of the conducting polymer film. For example,polypyrrole exhibits a conductivity that varies over five orders ofmagnitude by merely changing the counterion of the electrolyte.

Depending upon the desired physical and electrical properties of thepolymer, several supporting electrolytes can be used according to themethod of the present invention. The cation of the supportingelectrolyte is most preferably a tetraalkylammonium ion, with the alkylgroups having from one to ten carbon atoms. Typical examples includetetraethylammonium and tetrabutylammonium. These tetraalkylammonium ionsare especially useful because they are soluble in aprotic solvents andare highly dissociated in solution. For similar reasons, lithium isoften used as the cation of the supporting electrolyte.

The anion of the supporting electrolyte can be any anion that isessentially non-nucleophilic and that is not easily oxidized. Unsuitableanions, because of excessive nucleophilicity or facile oxidation,include the halides, hydroxyl, alkoxys, cyanide, acetate and benzoate.However, most other anions, organic and inorganic in structure, can beused in the electrochemical synthesis of the conducting polymers of thepresent invention. Suitable anions include tetrafluoroborate (BF₄ ⁻),perchlorate (ClO₄ ⁻), tetrachloroferrate III (FeCl₄ ⁻),tetrachlororuthenate (RuCl₄ ⁻), p-toluenesulfonate, picryl sulfonate,hexafluoroarsenate, trifluoromethylsulfonate (CF₃ SO₃ ⁻),hexafluorophosphate (PF₆ ⁻), fluorosulfonate, trifluoroacetate (CF₃ CO₂⁻), p-bromobenzenesulfonate, and perruthenate (RuO₄ ⁻²). Similarly,metals such as iron, cobalt, nickel, ruthenium, rhodium, platinum,osmium, iridium and palladium, when positioned as the central atom of ananion, like cobalt in porphyrin or iron in iron phthalocyanin, can beused as the anion of the supporting electrolyte.

Several enzymatically-based sensor systems involve the generation ofhydrogen peroxide as a secondary product. An example of such a system isthe detection of glucose using glucose oxidase. To achieve the fulladvantage of the analyte/probe systems of the present invention,tetrachlororuthenate (RuCl₄ ⁻) is used as the anion of the supportingelectrolyte. This ruthenium-based anion, or similar iron-based anions,serve not only as a compensating dopant for the polymer but also ascatalyst for the oxidative decomposition of hydrogen peroxide. Theimportance of this catalytic effect will be discussed more fullyhereinafter in regard to the detection of a specific analyte, glucose.

In addition to glucose oxidase, oxidase enzymes that employ oxygen as amediator, and therefore produce hydrogen peroxide include:

glucose oxidase,

cholesterol oxidase,

aryl-alchol oxidase,

L-gulonolactone oxidase,

galactose oxidase,

pyranose oxidase,

L-sorbase oxidase,

pyridoxin 4-oxidase,

alcohol oxidase,

L-2-hydroxyacid oxidase,

pyruvate oxidase,

oxalate oxidase,

glyoxylate oxidase,

dihydro-orotate oxidase,

lathosterol oxidase,

sarcosine oxidase,

N-methylamino-acid oxidase,

N⁶ -methyl-lysine oxidase,

6-hydroxyl-L-nicotine oxidase,

6-hydroxy-D-nicotine oxidase,

nitroethane oxidase,

sulphite oxidase,

thiol oxidase,

cytochrome c oxidase,

Pseudomonas cytochrome oxidase,

ascorbate oxidase,

o-aminophenol oxidase, and

3-hydroxyanthranilate oxidase.

As a result, in accordance with the methods of the present invention,analyte sensors can be made utilizing any oxidase enzyme by employingthe same method described above for the glucose oxidase embodiment.

In the electrochemical polymerization of the monomers of the presentinvention, a voltage of 0.8 was found to be a useful general value.However, this voltage is not necessarily an optimal value because thethreshold voltage for a majority of the monomers used to synthesizeconducting polymers is considerably below 0.8 volts.

The conductivity of the polymer films grown from the substituted2,5-di(2-thienyl)pyrrole monomers generally varied between 10⁻³ S/cm andapproximately 0.05 S/cm, as measured using an Alessi Industries fourpoint probe at a constant current of 15.0uA (microamperes). Films havinga conductivity of less than 1×10⁻² S/cm required the use of a lowerconstant current.

This relatively high conductivity is important because the conductivityof the polymer film is directly related to the conductivity of thediagnostic device. In particular, it was found that polymer films grownfrom the trifluoroacetyl blocked amines of structure (XIX) exhibited thehighest conductivity. The conductivities observed for the polymer filmsgenerated from the substituted 2,5-di(2-thienyl)pyrrole monomers weresignificantly higher than the conductivity of any derivatized polymerfilms reported in the prior art. Moreover, the stability of theconducting polymers generated from the substituted2,5-di(2-thienyl)pyrrole monomers is significantly improved incomparison to the prior art derivatized films.

In general, the morphology and electrical properties of the conductingpolymer depends upon the monomer, the supporting electrolyte and thepolymer film thickness. For example, the following procedure illustratesthe general preparation of a conducting polymer film from thedithienylpyrrole derivative,3-N-trifluoroacetamidomethyl-2,5-dithienylpyrrole (XIX). Thepolymerization of this monomer, and the other 3-substituted2,5-di(2-thienylpyrrole) derivatives, was performed in close analogy tothe electrochemical polymerization methods taught in the prior art.

More particularly, to electropolymerize3-N-trifluoroacetamidomethyl-2,5-dithienylpyrrole (XIX), a 30 mLsolution that is 5.0×10⁻³ M in the monomer (XIX) and 0.01M intetraethylammonium tetrachlororuthenate (C₂ H₅)₄ NRuCl₄ in acetonitrile(CH₃ CN) is prepared.

In addition, gold anodes were prepared sputtering approximately 1000 Å(Angstroms) of gold onto an appropriate substrate. Suitable substratesinclude teflon, chrome-treated glass, glass or polystyrene, with thesubstrate choice depending upon subsequent processing of the conductingfilm. A prescribed polymer growth area on the anode is defined byscreening a 2.25 cm² pattern. A single cell electrochemical apparatus,consisting of the above-defined anode, a reference electrode of Ag/Ag⁺in CH₃ CN, and a cathode, generally either a platinized titanium mesh orgold sputtered onto abraded glass is used. The cathode has a surfacearea of approximately 6.5 cm². The anode and cathode are positioned inparallel and separated by a distance of 2 cm. Prior to polymersynthesis, the electrochemical cell is purged of air by bubbling argonthrough the solution. A blanket of argon is maintained over the solutionduring polymer growth.

The observed current varies slightly depending upon the particular2,5-di(2-thienyl)pyrrole monomer being polymerized. The variance incurrent is more pronounced if the reaction conditions are altered.However, under the reaction conditions of this general example, acurrent of approximately 300-400 uA cm² (microamperes per cm²) istypical. For most applications, 0.2930 Coul cm² (coulombs cm²) ofcurrent are allowed to pass before polymerization is halted. This amountof current corresponds to a conducting polymer thickness ofapproximately 7325 Å. After electrochemical growth, the conductingpolymer films are rinsed thoroughly in sequential acetonitrile baths,and then dried either under vacuum or under an inert argon gas stream.

Bulk conductivity measurements on the polymer film were made afterstripping the film from the gold anode by using an epoxy support. Toremove the film from the anode, a 0.1 mL aliquot of Master Bond UV14ultraviolet curable epoxy is applied to the outer surface of the polymerand cured for approximately 2 minutes under a Xenon (Xe) UV lamp. Theepoxy-supported film then is etched from the gold surface using GOLDETCHANT, TYPE TFA, a commercial product available from the TranseneCompany, Inc.

The conducting polymer films then are thoroughly washed either in wateror in water followed by acetonitrile. The conductivity of the polymerfilm is measured using a four-point probe (Alessi Industries) at aconstant current of 15.0 uA (microamperes). However, for polymer filmshaving conductivities less than 1×10⁻² S/cm, it may be necessary to useless current, such as from about 5 to about 10 uA.

Similarly, conducting polymer films were grown on two electrodemicrodevices by an essentially identical method to the method describedabove except for the following modifications. The electrode, having atotal exposed surface area of approximately 2.7 mm², is linked in serieswith a scavenger electrode in order to retain a constant anode area of2.25 cm². In addition, the films were grown to thickness of only about0.07 Coul/cm² to enhance sensitivity to surface effects.

In accordance with an important feature of the present invention, theconductivity of polymers synthesized from the derivatives of2,5-di(2-thienyl)pyrrole can be enhanced by copolymerization of the2,5-di(2-thienyl)pyrrole derivative with pyrrole or other unsubstitutedparent heteroaromatic monomers. For example, conducting polymer filmswere grown from an electrolytic solution containing both pyrrole and aderivatized monomer of structure (XIX). Although it is known that themonomer units comprising the polymer film do not identically reflect theratio of monomers in the electrolytic solution, it has been proven byinfrared, ESCA, conductivity and current versus voltage studies that thederivatized monomer (XIX) was included in the conducting copolymer film.

The initial successful attempt at electrochemical copolymerizationutilized a monomer mix of an ethyl ester derivative of pyrrole andpyrrole. As will be detailed more fully hereinafter, a copolymer of theN-trifluoroacetamidomethyl dithienylpyrrole derivative (XIX) and pyrroleintroduced a sufficient number of functional sites into the copolymer toallow efficient covalent bonding of an analyte specific probe moleculeor a bridging molecule to the polymer. The conductivity of the resultingcopolymer films and the number of functional sites present on thecopolymer films can be adjusted and regulated by altering the relativeconcentrations of monomers in solution. As a result, conductingcopolymer films possessing conductivities as high as 10 S/cm and havingthe capability to covalently bind enzymes have been synthesized.

Copolymer films are synthesized in a method analogous to the synthesisof homopolymer films. Generally, the total concentration of oxidizablemonomer is maintained at 5.0×10⁻³ M. For example, a 1/1 copolymer ofpyrrole and 3-trifluoroacetamidomethyl-2,5-dithienylpyrrole (XIX) isgrown from a solution that is 2.5×10⁻³ M in pyrrole and 2.5×10⁻³ M inmonomer (XIX). It has been confirmed using IR, ESCA, current versusvoltage experiments and enzyme coupling experiments that both monomersare incorporated into the polymer chain. The ratio of monomer unitsincluded in the copolymer chain was not precisely determined, however,the ratio is not equal to the ratio of the monomers in solution.

It was observed that copolymerizing the 3-acetyl derivative of2,5-di(2-thienyl)pyrrole (XXIV) with pyrrole, under the conditionsdescribed above, produced films having a conductivity range ob 2.05×10⁻³to 7.02×10⁻³ S/cm, depending upon the monomer ratio.

Normally, a protecting group, such as the trifluoroacetyl group, isplaced on the amine group in order to protect the reactive amine moietyduring the polymerization process. After polymerization, thetrifluoroacetyl group then is removed to allow the amine functionalityto react with the bridging molecule or analyte specific probe molecule.However, by varying polymerization conditions, it is possible todirectly copolymerize the 3-aminomethyl-2,5-dithienylpyrrole monomer(XVIII), absent the trifluoracetyl protecting group, with pyrrole. Inaccordance with this direct copolymerization method, the subsequentremoval of the blocking group is avoided, and the polymer can be reacteddirectly with the bridging or specific probe molecule.

In accordance with an important feature of the present invention, therelative amounts of the unsubstituted parent heteroaromatic compound andthe 3-substituted 2,5-di(2-thienyl)pyrrole monomer present in themonomer mixture depends upon several variables, including the relativereactivities of the two monomers, the desired conductivity of theconducting copolymer, the number of desired sites in the copolymer forcovalently bonding the probe molecule and the general physical andchemical characteristics of the copolymer such as stability,brittleness, solubility, and the like. These variables can be defined bythose skilled in the art after considering such factors as the monomersto be used, the analyte to be detected, the polymerization andpostpolymerization reaction conditions to be encountered, and theanalyte test conditions to be encountered.

In addition to discovering a novel class of monomers, the2,5-di(2-thienyl)pyrroles (XI) that possess substituents at thethree-position of the central ring and can be readily polymerizedelectrochemically to yield organic conducting polymers, it also has beenfound that the conducting organic polymers can undergopost-polymerization reactions on the three-position substituents inorder to covalently bond bridging molecules or probe molecules to theconducting polymer. By covalently bonding the probe molecule to theconducting polymer either directly or through a bridging molecule, theconducting polymer can be utilized in a diagnostic device as an analytesensor for the specific analyte that reacts with the probe molecule.

The demonstration that post-polymerization chemistry can be performed onthe 3-position substituents of the heteroaromatic ring is an importantfeature of the present invention. Analogous to the difficulties imposedby steric requirements in growing the conducting polymers, it also canbe expected that steric interactions may make the 3-position chemicalmoiety unavailable for postpolymerization chemistry. In accordance withthe present invention, the demonstration that probe molecules can becovalently attached to the chemical moieties on the surface of theconducting polymer is both new and unexpected.

It was demonstrated that the three-position substituent can undergopostpolymerization reaction by the reaction of3-acetyl-2,5-dithienylpyrrole (XXIV) with phenylhydrazine to yield thecorresponding hydrazone derivative. However, in the course of thisreaction, the phenylhydrazine also reduced the polymer and thereforedestroyed the conductivity of the polymer film.

Another demonstration of postpolymerization polymer surface reactivitywas observed in the copolymer films of the3-trifluoroacetamidomethyl-2,5-dithienylpyrrole (XIX). The copolymerfilm was produced electrochemically at 0.8 V from a solution that was2.5×10⁻³ M in pyrrole, 2.5×10⁻³ M in the dithienylpyrrole monomer (XIX),and 0.01M in RuCl₄ ⁻ counterion. Using a monomer ratio of 9:1pyrrole/dithienylpyrrole monomer (XIX) produced similar copolymers.After polymerization, the free amine moiety on the polymer surface wasexposed by chemically removing the trifluoroacetyl protecting groups,yielding the copolymerized 3-aminomethyl derivative of dithienylpyrrole(XVIII). Among the available methods of removing the trifluoroacetylblocking group, it was found that exposing the copolymer film to asolution of 1×10⁻² M sodium methoxide in methanol (NaOCH₃ /CH₃ OH) for16 hours at room temperature is preferred. The presence of the freeamine moiety on the polymer surface was verified by usingradioactive-labeled reactive markers, ESCA studies, and binding studies.

After removal of the trifluoroacetyl protecting group, glucose oxidasewas covalently attached to the exposed free amine moieties on theconducting polymer surface by utilizing one of several availablechemical reactions. For example, the glucose oxidase was covalentlybound to the free amine moieties on the conducting polymer usingdimethyl adipimidate dihydrochloride as a bifunctional coupling agent tolink the free amine moieties to the lysyl ε-amino groups of the glucoseoxidase. Successful covalent bonding of the glucose oxidase to the aminemoieties was accomplished by exposing the polymer to a solution of 80mg/mL of dimethyl adipimidate dihydrochloride in 0.25M potassiumbicarbonate at pH 10 and 37° C. for ten minutes, followed by minutesexposure to a solution of 0.7 mg/mL glucose oxidase at 37° C. It wasfound that approximately, 0.5×10⁻¹² to 1.0×10⁻¹² moles/cm² of enzyme wasbound to the polymer surface under these conditions. The adipimidatecoupling reaction showed that proteins can be covalently attached to thepolymer surface. However, other chemical techniques can also be used inorder to better retain the electrical properties of the polymer byavoiding the basic conditions required in the adipimidate technique.

For example, an alternate technique for covalently attaching the enzymeto the conducting polymer surface uses monomeric glutaraldehyde as thecoupling agent. An effective covalent enzyme coupling has been achievedover a wide range of experimental conditions, therefore demonstratingthe flexibility in this technique. Generally, the conditions used tocovalently bond the glutaraldehyde to the polymer surface include:activation of the polymer in a solution of 6% glutaraldehyde in 0.1 Iphosphate buffer at 37° C. for 24 hours, followed by enzyme coupling ina solution of 0.07 mg/mL glucose oxidase in 0.1 I phosphate buffer at37° C. for 24 hours.

Analytical investigations have differentiated between glucose oxidasethat is non-covalently bound and glucose oxidase that is covalentlybound to the polymer film. It has been found that the covalently-boundglucose oxidase is present on the polymer surface at approximately1×10⁻¹² to 1.5×10⁻¹² moles cm², a value that is consistent with thetheoretical value calculated for a monolayer of covalently attachedenzyme. The method and conditions utilized in the enzyme attachmentprocedure are more fully described in the following Example 16.

EXAMPLE XV Glucose Oxidase Attachment to a Conducting Polymer

The covalent attachment of a biological probe to a conducting polymersurface has been demonstrated by covalently bonding glucose oxidase tothe surface of a conducting copolymer film synthesized from pyrrole and3-trifluoroacetamidomethyl-2,5-dithienylpyrrole (XIX). Similar resultswere obtained using copolymers obtained from monomer mixtures containing1/1 and 9/1 ratios of monomers (pyrrole/dithienylpyrrole (XIX)).

After copolymer synthesis, the copolymer film was divided in half. Onehalf of the film was coupled with the enzyme and the other half servedas a control. Both halves of the copolymer film were exposed to asolution of 1.0×10⁻² M sodium methoxide (NaOCH₃) in methanol at roomtemperature for approximately 16 hours to remove the trifluoroacetylblocking group and expose the free amine moieties on the copolymersurface. The two films then were washed twice with methanol and twicewith 0.1 ionic strength phosphate buffer of pH 7.

It is preferred that glutaraldehyde, the bridging molecule used tocouple the copolymer surface amine moieties to the ε-amino groups oflysine residues in the enzyme, is monomeric. The presence of monomericglutaraldehyde can be easily monitored by using UV spectroscopy becausepolymeric glutaraldehyde exhibits an intense peak at approximately 235nm (nanometers).

The preferred activation conditions include exposure of the copolymerfilms to a solution of 6% glutaraldehyde in phosphate buffer at 37° C.for 24 hours. The control half of the film is exposed only to thephosphate buffer and not the glutaraldehyde. Both films then were washedtwice with phosphate buffer. The preferred conditions for attachment ofthe glucose oxidase include exposure of the polymer film, activated withglutaraldehyde, to a solution containing 0.07 mg/mL of the enzyme inphosphate buffer at 37° C. for 24 hours. Both the glutaraldehyde treatedhalf of the polymer film and the control half of the polymer film areexposed to the enzyme solution. Both exposed films then are washedthoroughly in phosphate buffer followed by repeated, agitated washesover the course of several hours in 0.2M Tris buffer (0.25M NaCl) at pH8 containing 100 microliters of TRITON X-100 surfactant, available fromRohm and Haas Corp., Philadelphia, Pa., per 100 milliliters of buffer.

The amount of enzyme covalently bound to the polymer surface is assayedusing a Trinder reaction; wherein 28.5 mL of 2 mM3,5-dichloro-2-hydroxybenzenesulfonic acid disodium salt, 20 ug/mLhorseradish peroxidase, and 0.12M glucose in phosphate buffer is mixedwith 150 uL of 4 mM aminoantipyrene. The reaction of glucose oxidasewith glucose is kinetically monitored by assaying the resulting dye at520 nm. A series of solution phase standards was prepared simultaneouslyin order to quantify the enzyme attachment and to avoid a potential biasthat may arise if the activity of the bound enzyme is significantlydifferent than that of the solution phase enzyme.

Utilizing this enzyme attachment technique, enzymes have been covalentlybound to a number of polymer films under a wide range of conditions.Typically, using the coupling conditions described above on a 9/1copolymer film, the glutaraldehyde-treated half of the copolymer filmbound 0.99±0.31 pmol/cm² (picomoles/cm²) of enzyme, whereas the controlhalf of the copolymer film bound 0.65±0.13 pmol/cm² of enzyme. The 3/2ratio in the amount of enzyme coupled to the treated and untreatedhalves of the copolymer film did not conclusively prove that the enzymewas covalently bound to the copolymer film. However, extensiveanalytical studies showed that the 3/2 ratio actually does reflect thecovalent bonding of the enzyme to the glutaraldehyde. The analyticalstudies included aging properties, pH optimizing studies and pHstability studies.

The pH stability studies provided most convincing proof of covalentenzyme attachment to the bridging molecule. For example, a pH 10 (0.25MKHCO₃) wash of the copolymer films at 37° C. for 24 hours significantlydecreased the activity of the non-covalently attached enzyme on thecontrol portion of the film, but only slightly affected the activity ofthe covalently-bound enzyme on the glutaraldehyde treated portion of thefilm. Furthermore, the available evidence indicated that this observedeffect is due to washing, and not due to denaturing the enzyme.Similarly, discrimination ratios between glutaraldehyde-treated andcontrol films are commonly as high as 20 to 30/1 for films that havebeen washed in pH 10 potassium bicarbonate solution.

Although the previous example demonstrates the covalent bonding of anenzyme, glucose oxidase, to a bridging molecule, glutaraldehyde, that isin turn bonded covalently to a free amine moiety on the polymer surface,it is not necessary that the moiety on the polymer surface be limited tothe amine group. Similarly, the bridging molecule can be any moleculecapable of covalently bonding both to the reactive moiety on the polymersurface and to the analyte probe molecule. In addition, if possible, theprobe molecule can be bound directly to the surface of the polymerwithout utilizing a bridging molecule.

For example, depending upon the particular bridging molecule or theparticular probe molecule to be bound to the polymer surface, it may bemore advantageous to have a moiety other than a free aminomethyl grouppresent on the surface of the molecule. For instance, should a sulfidelinkage be desired, a thiol moiety may be introduced on the surface ofthe conducting polymer. Similarly, in addition to a free nitrogen orsulfur containing moiety on the polymer surface, other useful moietiesinclude those having an oxygen, such as hydroxyl groups; substitutedalkyl, such as halogen substituted alkyls; phosphorous containinggroups, such as phosphate; and other such moieties having reactivecenters, like carbonyl-containing groups or leaving groups, that canreact, covalently, with the bridging or probe molecule by addition orsubstitution reaction mechanisms.

Similarly, the bridging molecules can be any molecule that cancovalently bond both to the reactive moiety on the polymer surface andto the probe molecule. The size and chemical structure of the bridgingmolecule determines the rigidity or flexibility of the link between theprobe molecule and conducting polymer, and determines the distance theprobe molecule is positioned from the conducting polymer. Theflexibility of the bridging arm, and the distance between the probemolecule and conducting polymer surface, can have an effect on theability of the conducting polymer to sense the analyte in solution. Forexample, other representative bridging molecules, in addition toglutaraldehyde, include dialdehydes, such as glyoxal, malondialdehyde,succinaldehyde, and adipinaldehyde; and diamines, such as1,8-octanediamine, 4-aminomethyl-1,8-octanediamine and hexamethylenediamine. In addition to these examples, several other homo-andheterobifunctional spacer arms for the attachment of antibodies,proteins and specific binding sites to the 3-aminomethyldithienylpyrrole(XVIII) are known and are commercially available. These spacer arms aredescribed in the following publications:

Peters, K. and Richards, F. M. (1977) Ann. Rev. Biochem. 46, 523-551;

Freedman, R. B. (1979) Trends in Biochemical Sciences, September,193-197;

Das, M. and Fox, C. F. (1979) Ann. Rev. Biophys. Bioeng. 8, 165-193;

Ji, T. H. (1979) Biochim. Biophys. Acta 559, 39-69; and

Conn. M. (1983) in Methods in Enzymology 103, 49-58.

The majority of these bifunctional spacer arms react first with theamino group of the dithienylpyrrole (XVIII), and then can be selectivelyactivated or can react with the amino, sulfhydryl, or other reactivegroup of the antibody, protein or other probe molecule.

In addition, if permitted by steric interactions and it is so desired,the probe molecule can be covalently bound directly to the conductingpolymer surface. Similarly, the bridging molecule can be incorporatedinto the monomer, as exemplified in monomers XXVI and XXVII. In anyevent, the presence or absence, type, and size of the bridging moleculewill depend upon the nature of the reactive moiety present on thepolymer, the nature of the available reactive site on the probemolecule, steric interactions involving the polymer, probe molecule andbridging molecule, and the desired chemical and physical properties ofthe overall analyte sensing system.

In addition to a novel class of substituted monomers that yieldconducting polymers capable of postpolymerization covalent attachment ofanalyte specific probe molecules, and in accordance with anotherimportant feature of the present invention, the presence andconcentration of the specific analyte capable of reacting with the probemolecule can be determined. The presence of the analyte and/or itsconcentration in liquid media can be directly determined because theconductivity of the conducting polymer is altered by the interaction ofthe probe molecule with the analyte. This measurable electrical effectis detected either through a direct coupling of vibrational interactionsarising from the probe molecule-analyte reaction to the conductivity ofthe polymer or through conductivity changes resulting from secondaryeffects produced by probe molecule-analyte reaction products.

For the analyte to be detected through a direct coupling of thevibrational energy of the probe/analyte interaction to thephononassisted bipolaron transport of the polymer, the probe moleculemust be covalently bound to the conducting polymer surface. Aspreviously defined and as used throughout the specification, a phonon isdefined as a quantized, delocalized vibrational or elastic state of thelattice. It is theorized that phonons in conducting polymers are farmore localized and molecular in nature than phonons in metals. However,a phonon in conducting polymers is nevertheless delocalized over severalmonomer units.

As also discussed previously, a bipolaron is the charge carrier inheteroaromatic polymers. A bipolaron is a double charged, localizeddefect that confines a region of conducting polymer having a stabilizedquinoid-like character. The bipolaron is formed through the interactionof two polarons. A bipolaron is illustrated schematically for a genericheteroaromatic polymer in structure (III). Analytical evidence suggeststhat the bipolaron defect in structure (III) extends over about four toabout six monomer units.

In addition, the direct covalent bonding of the probe molecule to thepolymer facilitates the electrical detection of the change in polymerconductivity that involve the chemical effects of a secondary speciesgenerated by the analyteprobe molecule interaction upon the polymer. Thecovalent bond between the conducting polymer and the probe moleculeincreases efficiency by providing a high surface concentration of thesecondary reaction product.

As previously discussed, the general class of probe molecules includesproteins that are receptors. Examples of probe molecules includeenzymes, antigens, and ion-specific binding sites, like crown ethers.However, other probe molecules can be utilized in the method of thepresent invention to detect antigens, antibodies, haptens, enzymes,enzyme substrates, enzyme substrate analogs, agglutinins, lectin, enzymecofactors, enzyme inhibitors, hormones, and like analytes in liquidmedia. For each analyte, the analyte detection mechanism via theconducting polymer includes a direct observation of an enzyme/substrateor antigen/antibody reaction through the vibrational energy generatedfrom these reactions.

For example, the vibrational excitations induced in the probe moleculeby a probe molecule/analyte reaction can be transported through theprobe molecule in a localized waveform termed a soliton. This localizedenergy then can be transmitted to the phonon modes of the conductingpolymer by proper selection of the length and stiffness of the molecularcoupling arm, i.e., the bridging molecule, between the probe moleculeand the polymer. Since the electrical properties of doped heteroaromaticconducting polymers depend upon the excitations of the internalvibrational states of the probe molecule/analyte reactions, theconductivity of the polymer can thereby be directly modulated.

The prior art references regarding the transport of vibrational energyin proteins do not suggest using vibrational energy transport processesas a method of detecting presence and/or concentration of a specificanalyte. To date, the principal applications of vibrational energytransport have been in the development of models for the action ofmuscles.

Therefore, in accordance with the method of the present invention, thereaction between the probe molecule, such as an enzyme, and the analyteproduces vibrational interactions that pass through the probe molecule,and the bridging molecule if present, in a stable, pulse-like excitationknown as a soliton. In accordance with an important feature of thepresent invention, the energy of the vibrational interaction can pass asa soliton through the probe molecule and bridging molecule to theconducting polymer. Therefore, it is the efficient transmission of thevibrational energy to the polymer that affects its phonon-assistedbipolaron and produces a change in polymer conductivity. Theconductivity change of the polymer then is related to the amount ofanalyte in solution.

It is an important feature of the present invention that if the solitonmechanism cannot function, the vibrational energy arising from the probemolecule/analyte reaction would be dissipated before reaching theconducting polymer, and therefore preclude analyte concentrationdeterminations. Solitons result from a non-linear coupling between thevibrational excitation caused by an enzyme/substrate reaction and theresulting deformation in the protein structure caused by the generationof the vibrational excitation.

Soliton transport has been proposed as the mechanism for the usefultransport of the energy released during adenosine triphosphate (ATP)hydrolysis. A soliton avoids the thermal dispersion of most localizedvibrations by coupling the local vibrations to elastic waves of the hostpolymers. As a result, a localized energy pulse can be transported overlong distances. It is essential that the coupling of this travelingpulse to the polymer phonon modes be effected by a covalent link,otherwise, reflection of the soliton, and dispersion mediated byintervening solvent, severely diminishes the signal.

Solitons are launched only by chemical reactions, and not by the actionof heat or light. In addition, solitons will form only under a strongcoupling of the internal vibrations of the molecule with a localdeformation of the molecule. Therefore, in order to transport thevibrational energy induced by the chemical reaction via a soliton, themolecule must be sufficiently flexible such that it will deform. Thisdeformation can occur in soft chains, like proteins, and serves totransfer energy between different portions of the molecule. In general,a soliton is analogous to a tsunami, or a wave of water that coversextremely long distances without dissipation. The movement of electronsthrough a superconducting metal is another analogous transmission.Therefore, although a soliton is a wave, its stability allows a solitonto be regarded as particle-like.

In accordance with the method of the present invention, the vibrationalexcitation caused by the probe molecule/analyte reaction and theresulting molecular deformations balance each other, whereby thevibrational excitation moves through the protein uninhibited. Forexample, the alpha-helix structure, common in proteins, has thenecessary three-dimensional structure that allows a vibrationalexcitation at one end of the molecule to be transported to the other endof the molecule via a soliton. The alpha-helical proteins possess thecorrect chemical makeup and stereochemistry to self focus, or trap, thevibrational energy in the stable, pulse-like solitons, to yield anefficient and focused transport of energy.

Therefore, in accordance with the method of the present invention, thevibrational energy created by the reaction between the probe molecule,like a protein, and the analyte will pass to the bridging molecule.Additionally, by the proper selection of the bridging molecule, suchthat it essentially matches the flexibility, helical structure, hydrogenbonding and/or other chemical and physical characteristics of the probemolecule, the vibrational energy can pass through the bridging moleculereach the conducting polymer to measurably alter the conductivity of thepolymer.

In order to generate a soliton, it is essential that the molecule is nottoo rigid, that the molecule possesses a significant vibrational dipole,and that the molecule possesses sufficient mass. The solitons launcheddue to the vibrational energy induced in the peptide group by a chemicalreaction, can transfer energy along the alpha-helical protein moleculewithout transformation of the energy into disordered heat motion.

An example of transmitting vibrational energy through a molecule suchthat observable effects are seen in an area of the molecule relativelyfar from the reaction site is described by J. Schlessinger et al inProc. Nat. Acad. Sci. USA, 72, 2, 2775-2779 (1975). The authorsinvestigated antibody molecules, known to have a relatively largecentral fragment, termed F_(c), and outer fragments, termed F_(ab). Eachof these fragments is a protein, with the F_(ab) fragment bonded to theF_(c) fragment by a disulfide (--S--S--) linkage. The investigatorsfound that the vibrational energy, resulting from a reaction of anantigen with the F_(ab) fragment, can pass undiminished through theF_(ab) fragment and the disulfide linkage to cause a conformation changein the distant F_(c) fragment. It was also found that if the flexibledisulfide linkage was replaced with a rigid linkage, the vibrationalenergy could not be transported into the F_(c) antibody fragment. Thisenergy transfer mechanism is analogous to the method of the presentinvention, wherein the probe molecule and bridging molecule are designedto act as the F_(ab) and disulfide linkage in the antibody and deliverthe vibrational energy to the conducting polymer.

The effective transport of vibrational energy in an antibody can beinterpreted in a fashion that is distinct but complementary to thesoliton description. Following the arguments of Chou in Biopolymers,26:285 (1981), the transport from the F_(ab) portion of the antibody tothe F_(c) portion of the antibody may reflect a resonance interactioninvolving low frequency vibrations that incorporate large fractions ofthe molecule. The requirements for transport of such energy arefundamentally the same as those involved in coupling a soliton to thepolymer. The invention described herein would also serve to coupleresonant, low frequency vibrations to the polymer phonon modes.

Therefore, it is possible to detect the presence and concentration of aspecific analyte in solution by measuring the conductivity change in theconducting polymer resulting from the vibrational energy of the probemolecule-analyte reaction and transported by a soliton. For example, theF_(ab) fragment of an antibody can be covalently bound, through adisulfide linkage or a helical structure, to a conducting polymer. Thesubsequent reaction between a specific antigen and the F_(ab) fragmentof the antibody launches a soliton to transport the vibrational energyof the reaction to the conducting polymer. The transferred vibrationalenergy alters the conductivity of the conducting polymer and thereforeallows detection and measurement of the antigen. Furthermore, it is notessential that all of the vibrational energy be transported to theconducting polymer because it has been calculated that a loss of 70% ofthe vibrational energy nevertheless generates a 10% change inconductivity.

The antigen detection mechanism of the present invention is especiallyuseful because direct monitoring of antibody-antigen reactions by theprior art methods has proven very inefficient. Therefore, in accordancewith an important feature of the present invention, the antibody-antigenreaction can be detected and monitored via on-off fluctuationspectroscopy. This particular technique can measure the noise generatedby the antigen-antibody reaction. Each antigen-antibody reaction createsa vibrational energy pulse that travels through the probe molecule via asoliton to cause a temporary change in the conductivity of the polymer.By measuring this noise, i.e., the change in conductivity of theconducting polymer, and by determining the number of conductivity spikesgenerated, the presence and amount of a specific antigen can bedetermined.

In accordance with another important feature of the present invention,the transducing of the probe molecule/analyte interaction into anelectrical signal within the conducting polymer also can beaccomplished, or enhanced, by a secondary process. For instance, ammoniaaffects the conductivity of polypyrrole, therefore permitting thedetection of ammonia because as ammonia concentration increases,polypyrrole conductivity decreases. In the method of the presentinvention, the detection of a reaction product of an enzyme-substratereaction can be accomplished either through direct compensation of thedopant counterion or more reversibly by selecting a counterion polymerdopant that also serves as a catalyst for the secondary reaction. Forexample, tetrachlororuthenate (RuCl₄ ⁻) or tetrachloroferrate (III)(FeCl₄ ⁻) can act as a dopant-catalyst for the oxidation of hydrogenperoxide. For example, since hydrogen peroxide is generated in thereaction of glucose oxidase with glucose in the presence of oxygen, amethod for determining glucose concentrations in solutions is available.Although the use of a dopant-catalyst as an electrical transducer inheteroaromatic polymers is fully taught in U.S. Pat. No. 4,560,534, inaccordance with the method of the present invention, the ability tocovalently bond the enzyme to the conducting polymer surfacesignificantly enhances the effectiveness of the transduction mechanismby ensuring a high local surface concentration of the peroxide.

In general, it has been found that the method of the present inventioncan be used to detect the presence and concentration of a specificanalyte in liquid media. In addition, the detection data show that themechanism of analyte detection involves both the primary effect ofvibrational coupling between the probe molecule and the polymer and asecondary effect produced by the supporting electrolyte counterion onthe reaction product of the probe molecule-analyte reaction.

In particular, a microelectrode device consisting of an interdigitedpair of gold electrodes with an insulating spacing of 25 u (microns)served as a template for the analyte sensor. Thetrifluoroacetamidomethyl derivative of dithienylpyrrole (XIX) andpyrrole were electrochemically polymerized under previously describedconditions to yield a conducting copolymer film of approximately 1800 Åthickness. The copolymer bridged the insulating gap, thereby coveringthe entire device with copolymer film. Although the uniformity of thefilm thickness was not monitored, it was determined that the copolymerfilm was thinnest above the insulating regions of the template. Afterremoving the trifluoroacetyl protecting group, glucose oxidase wasattached to the conducting copolymer film using the dimethyl adipimidateprocedure discussed previously. The microelectrode devices then weremounted in a flow-through cell and exposed to varying concentrations ofhydrogen in buffer as well as 1000 mg/dL samples of D- and L-glucose.

An approximately linear dose response to hydrogen peroxide was observedover the concentration range of 0.044-0.88 mM. In addition, a D-glucoseresponse was observed that was approximately comparable to the hydrogenperoxide response at 0.44 mM. Significantly, no response to L-glucosewas observed therefore demonstrating that the sensitivity to D-glucoseactually was induced enzymatically.

The magnitude of the response also is significant. By assuming adiffusion rate for hydrogen peroxide of approximately 6×10⁻⁶ cm² /secand a surface coverage of approximately 0.6 pmol/cm², it can be shownthat a local concentration of 0.4 mM hydrogen peroxide cannot besustained because the diffusion rate of the hydrogen peroxide from theconducting copolymer film would far exceed the generation rate ofhydrogen peroxide. Therefore, the response of the microelectrode deviceto the glucose involved more than merely the enzymatic production ofhydrogen peroxide. As a result, indirect evidence exists for thevibrational coupling mechanism between the enzyme/substrate reaction andthe conducting polymer. This analyte detection mechanism is bothsurprising and unexpected, and is not suggested in the prior art.

In regard to hydrogen peroxide generation, the amount of enzymecovalently bound to the conducting copolymer film is not sufficient toproduce and maintain a significant macroscopic hydrogen peroxidecoverage on the electrode. As a result, over a small region (like 50 Å),the hydrogen peroxide concentration must decrease from the local surfacevalue to approximately zero. If the local surface value is assumed to be0.5 mM (5×10⁻⁷ mol/cm³), then the flux (J) of hydrogen peroxide awayfrom the 50 Å surface region (J=DΔC/L, wherein J is the flux, D is thediffusion coefficient (6×10⁻⁶ cm² /sec), C is the concentration drop(5×10⁻⁷ moles/cm³) and L is the distance over which the concentrationdrops (5×10⁻⁷ cm), is approximately 6×10⁻⁶ mol/sec/cm². The generationrate of hydrogen peroxide is determined by the surface concentration ofthe enzyme on the conducting copolymer. Assuming an activity of 20units/mg, a production rate for hydrogen peroxide of 1.192×10⁻¹²moles/sec/cm² is calculated. Clearly, the production rate for hydrogenperoxide cannot compete with the diffusion rate, therefore precluding alocal hydrogen peroxide concentration of 0.5 mM.

The primary features relating to the method of the present inventionhave been repeatedly observed. The new and unexpected results arisingfrom the method of the present invention will result in diagnosticdevices designed to assay liquid media for specific analytes.

From the foregoing, it is seen that the present invention is welladapted to attain all of the objects hereinabove set forth, togetherwith other advantages that are obvious and are inherent to the analytedetection system. The invention has the advantages of convenience,simplicity, relative economy, positiveness, effectiveness, durability,accuracy and directness of action. Among the advantages of the presentinvention is that the method operate nonoptically, can be constructed atrelatively low cost, have a great degree of flexibility with respect toformat, and can be constructed to have a relatively small size.

Although the present invention is primarily directed to assaying liquidmedia for various clinically significant substances or constituents inbiological fluids, such as urine and blood, including lysed or unlysedblood, blood plasma, blood serum, it should be understood that themethod of the present invention can be utilized for the detection ofnonbiological fluids, including swimming pool water, wines, etc.

It will be understood that the present disclosure has been made only byway of preferred embodiment and that numerous changes in details ofconstruction, combination, and arrangement of parts can be resorted towithout departing from the spirit and scope of the invention ashereunder claimed.

We claim:
 1. Substituted bithiophenes and dithienylpyrrolescharacterized by the formulae: ##STR28## wherein R isN-trifluoroacetamidomethyl, 2-hydroxyethyl, 2-phthalimidoethyl,2-trifluoroacetamidoethyl, acetyl, carboxyethyl, carboethoxyethyl,carbomethoxyethyl, (N-3-carbomethoxypropionyl)aminoethyl,(N-3-carboxypropionyl)amiomethyl, 2-methyldithioethyl,(N-imidazocarbonyl)amidomethyl, (4-nitrophenylcarbamoyl)amidomethyl,formylmethyl or carboxymethyl and R' is cyano, aminomethyl,N-trifluoroacetamidomethyl, 2-hydroxyethyl, 2-phthalimidoethyl,2-aminoethyl, 2-trifluoroacetamidoethyl, acetyl, carboxyethyl,carboethoxyethyl, carbomethoxyethyl,(N-3-carbomethoxypropionyl)aminoethyl,(N-3-carboxypropionyl)aminomethyl, 2-methyldithioethyl,(N-imidazocarbonyl)amidomethyl, (4-nitrophenylcarbamoyl)amidomethyl,formylmethyl or carboxymethyl.
 2. The bithiophenes and dithienylpyrrolesof claim 1 wherein R and R' are 2-trifluoroacetamidoethyl.
 3. Thedithienylpyrroles of claim 1 wherein R' is cyano, aminomethyl or2-aminoethyl.